CN105219829B - A method of preparing 9 Alpha-hydroxies-androstane -1,4- diene -3,17- diketone - Google Patents
A method of preparing 9 Alpha-hydroxies-androstane -1,4- diene -3,17- diketone Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of methods for preparing 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione to utilize mycobacteria using phytosterol as substrateMycobacterium(CICC 21097) and rhodococcus rhodochrousRhodococcus rhodochrous(CGMCC 4.1480) two kinds of microorganism mixed fungus fermentations prepare 9 Alpha-hydroxies-androstane -1,4- diene -3,17- diketone.The present invention also passes through adds compound chaotropic agent in the fermentation medium, and phytosterol is made to reach 84.3% to the highest molar yield of 9 α-OH-ADD.The purification step for reducing the method for two-step method preparation 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione simultaneously, reduces the usage amount of solvent.
Description
Technical field
The invention belongs to field of microbial pharmacy, are related to a kind of 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione, specifically
For be a kind of method using mixed bacterium bioconversion phytosterol preparation 9 Alpha-hydroxies-androstane -1,4- diene -3,17- diketone.
Background technique
Steroidal compounds (Steroids) are also known as steroids, are a kind of compounds containing cyclopentanoperhy drophenanthrene parent nucleus.Its
Basic structure is as follows, is made of three hexatomic rings and a five-membered ring, is referred to as A, B, C, D ring, generally in steroidal mother
There is angular methyl (- CH on the 10th and 13 of core3), the 3rd, 9,11 may have hydroxyl (- OH) or ketone group (- C=O), A ring or B
Ring is there are partial double bond, the side chain that has length different on the 17th.Due to substituent group, position of double bond or three-dimensional structure on steroidal parent nucleus
The difference of type etc. forms a series of compounds with unique physiological function.
Steroidal compounds general structure
Steroidal drug is the second major class drug that yield is only second to antibiotic in pharmaceuticals industry, and steroidal drug has anti-
The multiple efficacies such as allergy, Hemorrhagic shock, anti-inflammatory, antiallergic action are widely used in treating bronchial asthma, rheumatism joint
Inflammation, eczema, anaemia and the healing for promoting fracture and wound, also be used to treat the endocrine system diseases such as Ai Disenshi, to body
There is important role in terms of development, immunological regulation, treating skin disease and birth control.Steroidal drug is swollen to hormone-dependent type
The treatment of tumor has certain curative effect.With the rapid development of steroid chemical, steroid hormone class drug oneself become the weight of field of medicaments
Class is wanted, it is clinically very widely used.
Currently, the novel bacterial and new industrial research developing state about microbial method conversion steroid drugs are good.9α-OH-
Important source material and key intermediate of the ADD and 9 α-OH-ADD as synthesizing steroid class drug, the favor by more researcher.
Domestic and international research work is concentrated mainly in related fields prepares androstane-4-alkene-3,17- diketone using phytosterol
(4-AD) or Androsta-1,4-diene-3,17-dione (ADD), and with strains such as Nocard's bacillus or Rhodococcus sps to 4-AD or ADD
Carry out C9αHydroxylating prepares 9 Alpha-hydroxies-androstane-4-alkene-3,17- diketone (9 α-OH-AD) or 9 Alpha-hydroxies-androstane -1,4- diene -
3,17- diketone (9 α-OH-ADD).
(1) sterol prepares 4-AD or ADD
Microorganism for sterol side chain degradation mainly has mycobacteria (Mycobacterium), Nocard's bacillus
(Nocardia), arthrobacterium (Arthrobacterium), brevibacterium (Brevibacterium) etc..In numerous documents and patent
Display can all generate 4-AD and ADD, the generation mass ratio root of the two during carrying out Side chain cleavage using phytosterol simultaneously
There is different according to the difference of strain and production technology.As used bacillus amyloliquefaciens pair in 104403974 A of patent CN
Phytosterol carries out side chain cleavage, and the mass ratio that 4-AD and ADD are generated is 18:1；It is utilized in 1544618 A of patent CN
Mycobacterium sp.NRRL B 3683 carries out side chain excision to cholesterol, 4-AD the and ADD mass ratio of generation is 1:10.
Transformation time is generally in 100-120h.
(2) 4-AD or ADD prepares 9 α-OH-AD or 9 α-OH-ADD
Microorganism for the reaction of 9 'alpha '-hydroxylation of steroidal mainly has Rhodococcus sp (Rhodococcus), bar bacterium
(Corynebacterium), Nocard's bacillus (Nocardia) etc..As 103667126 A of patent CN describes a kind of utilization shame dirt
The method that mycobacteria prepares 9 α-OH-AD；A kind of utilization mycobacterium fortutitum preparation is disclosed in 103343155 A of patent CN
The method of 9 Alpha-hydroxy androstenedione.Transformation time is generally in 96-100h or so.
In conclusion preparing 9 α-OH-ADD from the point of view of with regard to current document and patent using phytosterol bio method, needing to lead to
Two-step fermentation is crossed to complete (i.e. two-step method), has no the document report for preparing 9 α-OH-ADD using phytosterol one-step method at present.
Two-step method generally converts ADD for phytosterol first with mycobacteria, then it is extracted, be refining to obtain ADD, recycle promise later
The bacterial strains such as Cattell bacterium or Rhodococcus sp carry out 9 'alpha '-hydroxylations to ADD and obtain 9 α-OH-ADD.But above-mentioned two-step method exists as follows not
Foot: when 1. phytosterol is converted to the direction ADD, the utilization efficiency of phytosterol is relatively low.What 2. Side chain cleavage fermentation process generated
Product is difficult to be isolated and purified containing 4-AD and ADD since 4-AD is close with the structure of ADD simultaneously.3. product
ADD later period extraction purification needs to extract purification using a large amount of suitable organic solvent, to obtain the higher ADD of purity,
It could be as the substrate of subsequent 9 'alpha '-hydroxylation.So the industrial biological method of exploitation more high efficiency and time conservation prepares 9 α-OH-
The method of ADD has very high application value and prospect.
Summary of the invention
For above-mentioned technical problem in the prior art, 9 Alpha-hydroxies-androstane-Isosorbide-5-Nitrae-two is prepared the present invention provides a kind of
The method of the method for alkene -3,17- diketone, this 9 Alpha-hydroxies of the preparation-Androsta-1,4-diene-3,17-dione solves
The method conversion ratio for preparing 9 Alpha-hydroxies-androstane -1,4- diene -3,17- diketone using two-step fermentation in the prior art is low, point
The technical issues of from purification difficult.
The present invention provides a kind of methods for preparing 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione, with phytosterol
For substrate, two kinds of micro- lifes of mycobacteria Mycobacterium and rhodococcus rhodochrous Rhodococcus rhodochrous are utilized
Object mixed fungus fermentation prepares 9 Alpha-hydroxies-androstane -1,4- diene -3,17- diketone.
Further, the method includes the following steps:
1) prepare fermentation medium containing phytosterol the step of；
2) compound chaotropic agent is added in the fermentation medium, the compound chaotropic agent is by ethyl alcohol and polyethylene glycol
Composition, in the fermentation medium, the concentration of the ethyl alcohol is 10.0-50.0g/L, the concentration of the polyethylene glycol
For 5.0-20.0g/L；
3) one is inoculated with mycobacteria Mycobacterium and rhodococcus rhodochrous Rhodococcus respectively
The step of rhodochrous, the inoculum concentration of the mycobacteria Mycobacterium are 10.0-15.0%, conversion culture 0-
After 48h, then 10.0-20.0% rhodococcus rhodochrous Rhodococcus rhodochrous being accessed, fermentation temperature is 28-32 DEG C,
Fermentation time is 72-120h, and fermentation medium pH is between 6.5-7.5；
4) 70-90 DEG C of fermentation liquid is inactivated after fermentation, is cooled to 20-30 DEG C by the step of product Hydrolysis kinetics
Afterwards, plate compression takes filter cake to be extracted, and using filter, decoloration, distillation, purification, obtains 9 Alpha-hydroxies-androstane-Isosorbide-5-Nitrae-diene-
3,17- diketone.Further, the formula of the fermentation medium is as follows: glucose 10.0-30.0g/L, ammonium dihydrogen phosphate 2.0-
4.0g/L, ferric citrate 0.03-0.08g/L, epsom salt 1.5-3.5g/L, dipotassium hydrogen phosphate 0.5-1.5g/L, phosphoric acid
Potassium dihydrogen 0.5-1.5g/L, beet molasses 5.0-9.0g/L, phytosterol 30.0-40.0g/L.
Further, the deposit number of the mycobacteria Mycobacterium is CICC 21097, comes from Chinese industrial
Microbiological Culture Collection administrative center；The deposit number of the rhodococcus rhodochrous Rhodococcus rhodochrous is
CGMCC 4.1480 comes from China General Microbiological culture presevation administrative center.
The present invention utilizes mycobacteria Mycobacterium (CICC 21097) and rosiness using phytosterol as substrate
Two kinds of microorganism mixed fungus fermentations of Rhodococcus sp Rhodococcus rhodochrous (CGMCC 4.1480) prepare 9 α-OH-ADD, also
By adding compound chaotropic agent in the fermentation medium, to phytosterol carry out bioconversion, make phytosterol Side chain cleavage with
C9αPosition one step of hydroxylating is completed, and phytosterol is made to reach 84.3% to the highest molar yield of 9 α-OH-ADD.
Method and two-step method of the invention (first makes phytosterol bio-conversion ADD, then using ADD as substrate, in its C9α
Position hydroxylating obtains 9 α-OH-ADD) compared to having the following advantages: (1) one-step method mix bacterium transformation phytosterin prepare 9 α-OH-ADDs,
Its hydroxylation procedures is more conducive to phytosterol and converts to the direction ADD, improves the utilization efficiency of phytosterol.(2) it reduces and mentions
Pure step eliminates the extraction purification process of ADD in two-step method, so that the purification of two steps is become a step, reduces solvent usage amount.(3)
The generation of by-product 4-AD and 9 α-OH-AD is reduced, biological transformation ratio of the phytosterol to 9 α-OH-ADD is improved, shortens biology hair
The ferment period.
The advantages of present invention between two bacterial strains using acting synergistically promotes phytosterol more to carry out to 9 α-OH-ADD
Conversion reduces the generation of by-product 4-AD and 9 α-OH-AD, improves biologicak efficiency of the phytosterol to 9 α-OH-ADD, shortens life
Object fermentation period largely reduces the effect of production cost.
The present invention is compared with prior art, and technological progress is significant.The present invention uses one-step conversion, reduces and mentions
Pure step eliminates the extraction purification process of ADD in two-step method, the purification of two steps is made to become a step, substantially reduces solvent use
Amount, reduces production cost.
Detailed description of the invention
Fig. 1 shows that mixed bacterium bioconversion phytosterol prepares the process of 9 α-OH-ADD.
Specific embodiment
The present invention is further described below by specific embodiment, but is not intended to limit the present invention.
The mycobacteria Mycobacterium (CICC 21097) that the present invention uses is the microorganism of public offering, is come from,
Chinese industrial Microbiological Culture Collection administrative center, the address of the mechanism are as follows: the institute 6 of Jiuxianqiao, Chaoyang District, Beijing City Road 24
Building (100015), can be bought by phone, can also be appointed by ordering site (http://www.china-cicc.org)
As long as who pays relevant expense and can buy.
The rhodococcus rhodochrous Rhodococcus rhodochrous (CGMCC 4.1480) that the present invention uses is open sells
The microorganism sold, comes from, China General Microbiological culture presevation administrative center, the address of the mechanism are as follows: Chaoyang District, Beijing City north
No. 3 Institute of Microorganism, Academia Sinica, institute of occasion West Road 1, can be bought by phone, can also pass through ordering site
(http://www.cgmcc.net), as long as anyone, which pays relevant expense, can buy.
Embodiment 1
Mycobacteria Mycobacterium (CICC 21097) and rhodococcus rhodochrous Rhodococcus
Rhodochrous (CGMCC 4.1480) obtains the required strain that ferments by inclined-plane culture and seed culture.One, branch
Bacillus Mycobacterium (CICC 21097) slant strains and seed liquor preparation
(1) inclined-plane culture
Slant medium: peptone 5.0g/L, beef extract 3.0g/L, sodium chloride 5.0g/L, glucose 10.0g/L, agar
20.0g/L is cultivated 5-7 days under the conditions of 29 DEG C.
(2) seed culture
Seed culture medium: glucose 15.0g/L, peptone 5.0g/L, beef extract 3.0g/L, sodium chloride 5.0g/L, seven water
Magnesium sulfate 2.5g/L, ammonium dihydrogen phosphate 3.5g/L, dipotassium hydrogen phosphate 1.0g/L, 7.0,121 DEG C of potassium dihydrogen phosphate 1.0g/L, pH
Moist heat sterilization 30min.
The bacterial strain that step (1) is cultivated aseptically is inoculated with 2 rings in equipped with 50mL seed culture medium with oese
In the shaking flask of 250mL, under the conditions of 30 DEG C, rotary shaker 200r/min cultivates 48h, and seed liquor is made.
Two, rhodococcus rhodochrous Rhodococcus rhodochrous (CGMCC 4.1480) slant strains and seed liquor
Preparation
(1) inclined-plane culture
Slant medium: glucose 10.0g/L, tryptone 15.0g/L, soy peptone 5.0g/L, sodium chloride 5.0g/
L, agar 20.0g/L are cultivated 5-6 days under the conditions of 30 DEG C.
(2) seed culture
Seed culture medium: glucose 10.0g/L, yeast extract 20.0g/L, fish peptone 5.0g/L, cornstarch 9.0g/L,
7.0,121 DEG C of moist heat sterilization 30min of dipotassium hydrogen phosphate 2.5g/L, pH.
The bacterial strain that step (1) is cultivated aseptically is inoculated with 1 ring in equipped with 50mL seed culture medium with oese
In the shaking flask of 250mL, under the conditions of 30 DEG C, rotary shaker 200r/min cultivates 30h, and seed liquor is made.
Three, fermented and cultured
Fermentation medium: phytosterol 35.0g/L, glucose 21.0g/L, ammonium dihydrogen phosphate 3.5g/L, ferric citrate
0.05g/L, epsom salt 2.0g/L, dipotassium hydrogen phosphate 1.2g/L, potassium dihydrogen phosphate 1.0g/L, beet molasses 6.0g/L, pH
7.0。
By the fermentor equipped with fermentation medium in 121 DEG C of high pressure steam sterilization 15min.
Compound chaotropic agent: ethyl alcohol 30.0g/L, polyethylene glycol 10.0g/L is initially added in culture in the medium.It is connect again
Kind, the inoculum concentration of mycobacteria Mycobacterium CICC (21097) is 12.0%, is inoculated with while mycobacteria inoculation
The inoculum concentration of rhodococcus rhodochrous, rhodococcus rhodochrous Rhodococcus rhodochrous (CGMCC 4.1480) is
17.0%, bioconversion is carried out using 5L fermentor, liquid amount 70%, ventilatory capacity 1.0v/vmin, temperature control is 30
DEG C, revolving speed 300r/min, every 12h sampling TLC monitoring during conversion.Fermented and cultured 96h, sampling TLC analysis, no plant steroid
Alcohol spot, conversion are basically completed.Sample is extracted with ethyl acetate, and supernatant is collected by centrifugation using on high-efficient liquid phase chromatogram technique analysis
The content for stating 9 α-OH-ADD in resulting fermentation liquid is 17.5g/L, and the content of 9 α-OH-AD is 0.25g/L, phytosterol mole
Conversion ratio is 72.4%.
After fermentation, 85 DEG C of fermentation liquid are inactivated, after being cooled to 25 DEG C, plate compression, filter cake is extracted with dichloromethane,
9 α-OH-ADD crude products are obtained using processes such as filter, decoloration and distillations, the mashing purification of crude product again with toluene finally obtains 9 α-
OH-ADD fine work.High-efficient liquid phase chromatogram technique analysis purity is 98.7%.
Embodiment 2
By mycobacteria Mycobacterium (CICC 21097) and rhodococcus rhodochrous Rhodococcus
Seed liquor (method is with embodiment 1) is made in rhodochrous (CGMCC 4.1480) bacterial strain, then carries out fermented and cultured.
Fermentation medium: phytosterol 35.0g/L, ethyl alcohol 31.0g/L, polyethylene glycol 9.0g/L, glucose 20.0g/L,
Ammonium dihydrogen phosphate 3.0g/L, ferric citrate 0.05g/L, epsom salt 2.5g/L, dipotassium hydrogen phosphate 1.0g/L, biphosphate
Potassium 1.0g/L, beet molasses 6.0g/L, pH 7.0.
By the fermentor equipped with fermentation medium in 121 DEG C of high pressure steam sterilization 15min.
Compound chaotropic agent: ethyl alcohol 31.0g/L, polyethylene glycol 9.0g/L is initially added in culture in the medium.It is connect again
Kind, the inoculum concentration of mycobacteria Mycobacterium CICC (21097) is 12.0%, after mycobacteria conversion culture for 24 hours
It is inoculated with rhodococcus rhodochrous, the inoculum concentration of rhodococcus rhodochrous Rhodococcus rhodochrous (CGMCC 4.1480) is
18.0%, bioconversion is carried out using 5L fermentor, liquid amount 70%, ventilatory capacity 1.0v/vmin, temperature control is 30
DEG C, revolving speed 300r/min, every 12h sampling TLC monitoring during conversion.Fermented and cultured 96h, sampling TLC analysis, no plant steroid
Alcohol spot, conversion are basically completed.Sample is extracted with ethyl acetate, and supernatant is collected by centrifugation using on high-efficient liquid phase chromatogram technique analysis
The content for stating 9 α-OH-ADD in resulting fermentation liquid is 20.4g/L, and the content of 9 α-OH-AD is 0.19g/L, phytosterol mole
Conversion ratio is 84.3%.
After fermentation, 85 DEG C of fermentation liquid are inactivated, after being cooled to 25 DEG C, plate compression, filter cake is extracted with dichloromethane,
9 α-OH-ADD crude products are obtained using processes such as filter, decoloration and distillations, the mashing purification of crude product again with toluene finally obtains 9 α-
OH-ADD fine work, high-efficient liquid phase chromatogram technique analysis purity are 99.1%.
Embodiment 3
Mycobacteria Mycobacterium (CICC 21097) and rhodococcus rhodochrous Rhodococcus
Seed liquor (method is with embodiment 1) is made in rhodochrous (CGMCC 4.1480) bacterial strain, then carries out fermented and cultured.
Fermentation medium: phytosterol 35.0g/L, glucose 22.0g/L, ammonium dihydrogen phosphate 3.0g/L, ferric citrate
0.05g/L, epsom salt 2.0g/L, dipotassium hydrogen phosphate 1.0g/L, potassium dihydrogen phosphate 1.0g/L, beet molasses 6.0g/L, pH
7.0。
By the fermentor equipped with fermentation medium in 121 DEG C of high pressure steam sterilization 15min.
Compound chaotropic agent: ethyl alcohol 30.0g/L, polyethylene glycol 9.0g/L is initially added in culture in the medium.It is connect again
Kind, the inoculum concentration of mycobacteria Mycobacterium CICC (21097) is 13.0%, after mycobacteria converts culture 48h
It is inoculated with rhodococcus rhodochrous, the inoculum concentration of rhodococcus rhodochrous Rhodococcus rhodochrous (CGMCC 4.1480) is
17.0%, bioconversion is carried out using 5L fermentor, liquid amount 70%, ventilatory capacity 1.0v/vmin, temperature control is 30
DEG C, revolving speed 300r/min, every 12h sampling TLC monitoring during conversion.Fermented and cultured 96h, sampling TLC analysis, no plant steroid
Alcohol spot, conversion are basically completed.Sample is extracted with ethyl acetate, and supernatant is collected by centrifugation using on high-efficient liquid phase chromatogram technique analysis
The content for stating 9 α-OH-ADD in resulting fermentation liquid is 18.3g/L, and the content of 9 α-OH-AD is 0.31g/L, phytosterol mole
Conversion ratio is 75.7%.
After fermentation, 85 DEG C of fermentation liquid are inactivated, after being cooled to 25 DEG C, plate compression, filter cake is extracted with dichloromethane,
9 α-OH-ADD crude products are obtained using processes such as filter, decoloration and distillations, the mashing purification of crude product again with toluene finally obtains 9 α-
OH-ADD fine work, high-efficient liquid phase chromatogram technique analysis purity are 98.6%.
Embodiment 4
Mycobacteria Mycobacterium (CICC 21097) and rhodococcus rhodochrous Rhodococcus
Seed liquor (method is with embodiment 1) is made in rhodochrous (CGMCC 4.1480) bacterial strain, then carries out fermented and cultured.
Fermentation medium: phytosterol 30.0g/L, glucose 10.0g/L, ammonium dihydrogen phosphate 2.0g/L, ferric citrate
0.08g/L, epsom salt 3.5g/L, dipotassium hydrogen phosphate 1.5g/L, potassium dihydrogen phosphate 1.5g/L, beet molasses 9.0g/L, pH
7.5。
By the fermentor equipped with fermentation medium in 121 DEG C of high pressure steam sterilization 15min.
Compound chaotropic agent: ethyl alcohol 10.0g/L, polyethylene glycol 5.0g/L is initially added in culture in the medium.It is connect again
Kind, the inoculum concentration of mycobacteria Mycobacterium CICC (21097) is 10.0%, after mycobacteria conversion culture for 24 hours
It is inoculated with rhodococcus rhodochrous, the inoculum concentration of rhodococcus rhodochrous Rhodococcus rhodochrous (CGMCC 4.1480) is
10.0%, bioconversion is carried out using 5L fermentor, liquid amount 70%, ventilatory capacity 1.0v/vmin, temperature control is 28
DEG C, revolving speed 300r/min, every 12h sampling TLC monitoring during conversion.Fermented and cultured 72h, sampling TLC analysis, no plant steroid
Alcohol spot, conversion are basically completed.Sample is extracted with ethyl acetate, and supernatant is collected by centrifugation using on high-efficient liquid phase chromatogram technique analysis
The content for stating 9 α-OH-ADD in resulting fermentation liquid is 17.1g/L, and the content of 9 α-OH-AD is 0.20g/L, phytosterol mole
Conversion ratio is 82.5%.
After fermentation, 85 DEG C of fermentation liquid are inactivated, after being cooled to 25 DEG C, plate compression, filter cake is extracted with dichloromethane,
9 α-OH-ADD crude products are obtained using processes such as filter, decoloration and distillations, the mashing purification of crude product again with toluene finally obtains 9 α-
OH-ADD fine work, high-efficient liquid phase chromatogram technique analysis purity are 98.8%.
Embodiment 5
Mycobacteria Mycobacterium (CICC 21097) and rhodococcus rhodochrous Rhodococcus
Seed liquor (method is with embodiment 1) is made in rhodochrous (CGMCC 4.1480) bacterial strain, then carries out fermented and cultured.
Fermentation medium: phytosterol 40.0g/L, glucose 30.0g/L, ammonium dihydrogen phosphate 4.0g/L, ferric citrate
0.03g/L, epsom salt 1.5g/L, dipotassium hydrogen phosphate 0.5g/L, potassium dihydrogen phosphate 0.5g/L, beet molasses 5.0g/L, pH
6.5。
By the fermentor equipped with fermentation medium in 121 DEG C of high pressure steam sterilization 15min.
Compound chaotropic agent: ethyl alcohol 50.0g/L, polyethylene glycol 20.0g/L is initially added in culture in the medium.It is connect again
Kind, the inoculum concentration of mycobacteria Mycobacterium CICC (21097) is 15.0%, after mycobacteria conversion culture for 24 hours
It is inoculated with rhodococcus rhodochrous, the inoculum concentration of rhodococcus rhodochrous Rhodococcus rhodochrous (CGMCC 4.1480) is
20.0%, bioconversion is carried out using 5L fermentor, liquid amount 70%, ventilatory capacity 1.0v/vmin, temperature control is 32
DEG C, revolving speed 300r/min, every 12h sampling TLC monitoring during conversion.Fermented and cultured 120h, sampling TLC analysis, no plant
Sterol spot, conversion are basically completed.Sample is extracted with ethyl acetate, and supernatant is collected by centrifugation using high-efficient liquid phase chromatogram technique analysis
The content of 9 α-OH-ADD is 21.5g/L in above-mentioned resulting fermentation liquid, and the content of 9 α-OH-AD is 0.24g/L, and phytosterol rubs
Your conversion ratio is 77.8%.
After fermentation, 85 DEG C of fermentation liquid are inactivated, after being cooled to 25 DEG C, plate compression, filter cake is extracted with dichloromethane,
9 α-OH-ADD crude products are obtained using processes such as filter, decoloration and distillations, the mashing purification of crude product again with toluene finally obtains 9 α-
OH-ADD fine work, high-efficient liquid phase chromatogram technique analysis purity are 98.1%.
Comparative example 1
Seed liquor (method is with embodiment 1) is made in mycobacteria Mycobacterium (CICC21097) bacterial strain, then into
Row fermented and cultured.
Fermentation medium is as described in Example 2, by the fermentor equipped with fermentation medium in 121 DEG C of high pressure steam sterilizations
15min。
Compound chaotropic agent: ethyl alcohol 31.0g/L, polyethylene glycol 9.0g/L is initially added in culture in the medium.It is connect again
Kind, the inoculum concentration of mycobacteria Mycobacterium (CICC 21097) is 12.0%, carries out biology using 5L fermentor and turns
Change, liquid amount 70%, ventilatory capacity 1.0v/vmin, temperature is controlled at 30 DEG C, revolving speed 300r/min, every during conversion
Every 12h sampling TLC monitoring.Fermented and cultured 96h, sampling TLC analysis, conversion are stagnated.Sample is extracted with ethyl acetate, and is collected by centrifugation
Supernatant uses the content of ADD in the above-mentioned resulting fermentation liquid of high-efficient liquid phase chromatogram technique analysis for 16.7g/L, and the content of 4-AD is
0.87g/L, phytosterol molar yield are 72.9%.
After fermentation, 85 DEG C of fermentation liquid are inactivated, after being cooled to 25 DEG C, plate compression, filter cake is extracted with dichloromethane,
ADD crude product is obtained using processes such as filter, decoloration and distillations, the mashing purification of crude product again with toluene finally obtains ADD fine work, high
Effect liquid phase chromatogram method purity assay is 98.5%.
Comparative example 2
Seed liquor (side is made in the bacterial strain of rhodococcus rhodochrous Rhodococcus rhodochrous (CGMCC 4.1480)
Method is with embodiment 1), then carry out fermented and cultured.
Fermentation medium is as described in Example 2, and substrate is changed into the ADD of 24.0g/L by the phytosterol of 35.0g/L.It will dress
There is the fermentor of fermentation medium in 121 DEG C of high pressure steam sterilization 15min.
Compound chaotropic agent: ethyl alcohol 31.0g/L, polyethylene glycol 9.0g/L is initially added in culture in the medium.It is connect again
Kind, the inoculum concentration of rhodococcus rhodochrous Rhodococcus rhodochrous (CGMCC 4.1480) is 18.0%, is sent out using 5L
Fermentation tank carries out bioconversion, and liquid amount 70%, ventilatory capacity 1.0v/vmin, temperature control is at 30 DEG C, revolving speed 300r/
Min, every 12h sampling TLC monitoring during conversion.Fermented and cultured 72h, sampling TLC analysis, no ADD spot convert substantially complete
At.Sample is extracted with ethyl acetate, and supernatant is collected by centrifugation using in the above-mentioned resulting fermentation liquid of high-efficient liquid phase chromatogram technique analysis 9
The content of α-OH-ADD is 22.6g/L, and ADD molar yield is 93.8%.
After fermentation, 85 DEG C of fermentation liquid are inactivated, after being cooled to 25 DEG C, plate compression, filter cake is extracted with dichloromethane,
9 α-OH-ADD crude products are obtained using processes such as filter, decoloration and distillations, the mashing purification of crude product again with toluene finally obtains 9 α-
OH-ADD fine work, high-efficient liquid phase chromatogram technique analysis purity are 99.1%.
By embodiment it is found that one-step method is compared with two-step method, one-step method is had the advantage that
1. helping to improve the transformation efficiency of phytosterol.As described in comparative example 1 and 2, two-step method bioconversion is planted
Object sterol prepares 9 α-OH-ADD, and phytosterol end molar yield is 72.9% × 93.8%=68.4%；And such as embodiment 2
Described, one-step method bioconversion phytosterol prepares 9 α-OH-ADD, and phytosterol molar yield is up to 84.3%.
2. helping to shorten fermentation period.As described in comparative example 1 and 2, the two-step method microorganism conversion time is about
168h, and it is as described in Example 2, and it is about 96h that one-step method mixed fermentation, which produces 9 α-OH-ADD bioconversion times, one-step method
Bioconversion time shortens about 72h than two-step method.
3. reducing solvent usage amount.One-step method eliminates the extraction purification process of ADD in two-step method, becomes the purification of two steps
One step reduces solvent usage, reduces production cost.
4. reducing the generation of Main By product 4-AD and 9 α-OH-AD.It can compared with comparative example 1 by embodiment 2
To find out, when carrying out single bacterium bioconversion to phytosterol using mycobacteria, while the generation of ADD and 4-AD are had, and two
The mass ratio of person is about 19:1.And when two kinds of bacterium mixed fermentations being utilized to produce 9 α-OH-ADD, while having 9 α-OH-ADD and 9 α-
The generation of OH-AD, and the mass ratio of the two is about 107:1.I.e. one-step method mix bacterium transformation phytosterin produce 9 α-OH-ADD can be with
The effective generation for reducing by-product 4-AD and 9 α-OH-AD, hydroxylation procedures are more conducive to phytosterol to the direction ADD
Conversion, improves the utilization efficiency of phytosterol.
Above content is only the basic explanation under present inventive concept, and any etc. made by technical solution according to the present invention
Effect transformation, is within the scope of protection of the invention.
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| CN108913643A (en) * | 2018-08-01 | 2018-11-30 | 天津科技大学 | A method of it improving mycobacteria regenerating coenzyme and androstenedione is promoted to produce simultaneously |
| CN111194825A (en) * | 2018-11-16 | 2020-05-26 | 中国科学院天津工业生物技术研究所 | A kind of harmless treatment method of steroid bacteria residue |
| CN109355345A (en) * | 2018-11-22 | 2019-02-19 | 安徽建筑大学 | A method for synthesizing androst-4-ene-3,7-dione by utilizing mycobacteria to degrade phytosterol |
| CN109652338B (en) * | 2019-01-24 | 2021-04-09 | 天津科技大学 | Mycobacterium fortuitum for high yield of 9 alpha-OH-AD and application thereof |
| CN114672427A (en) * | 2021-10-26 | 2022-06-28 | 郑州大学 | Mycobacterium for producing androstane-1, 4 diene-3, 17-dione and application thereof |
| CN114854636A (en) * | 2022-05-22 | 2022-08-05 | 郑州大学 | Method for producing androstane-1, 4-diene-3, 17-dione (ADD) by immobilizing mycobacterium |
| CN115537446A (en) * | 2022-09-06 | 2022-12-30 | 天津中医药大学 | Application of rhodococcus aetherivorans in production of 4-androstenedione by converting cholesterol |
| CN115433698A (en) * | 2022-10-18 | 2022-12-06 | 湖北共同药业股份有限公司 | A kind of mycobacterium smegmatis bacterial liquid and its preparation method and its application in the preparation of steroid drug intermediates |
| CN119931969B (en) * | 2024-12-25 | 2026-03-17 | 湖北共同甾体药物研究院有限公司 | A dehydrogenase and a method for synthesizing 11α-OH-ADD |
| CN120173832B (en) * | 2025-05-21 | 2025-09-23 | 沈阳博泰生物制药有限公司 | Mycobacterium neogoldensis and its use in preparing steroid drug intermediates |
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