CN105219829A - A kind of method preparing 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione - Google Patents
A kind of method preparing 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione Download PDFInfo
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Abstract
The invention discloses one and prepare 9 Alpha-hydroxies-androstane-1, 4-diene-3, the method of 17-diketone, take plant sterol as substrate, do you utilize mycobacterium <i>MycobacteriumLEssT.LTssT.L T/i>(CICC? 21097) and rhodococcus rhodochrous <i>Rhodococcus? rhodochrous</i>(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 by adding compound chaotropic agent in the fermentation medium, makes plant sterol reach 84.3% to the highest molar yield of 9 α-OH-ADD.Decrease the purification step that two-step approach prepares the method for 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione simultaneously, reduce the usage quantity of solvent.
Description
Technical field
The invention belongs to field of microbial pharmacy, relate to a kind of 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione, a kind ofly specifically adopt mixed bacterium bioconversion phytosterol to prepare the method for 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione.
Background technology
Steroidal compounds (Steroids), also known as steroid, is the compound that a class contains perhydrocyclopentanophenanthrene parent nucleus.Its basic structure is as follows, is made up of, is called A, B, C, D ring three six-rings and a five-ring, generally steroidal parent nucleus the 10th and 13 on have angular methyl(group) (-CH
3), the 3rd, 9,11 may have hydroxyl (-OH) or ketone group (-C=O), and A ring or B ring exist partial double bond, the side chain that the 17th has length different.Due to the difference of substituting group, position of double bond or steric configuration etc. on steroidal parent nucleus, define a series of compound with unique physiological function.
Steroidal compounds general structure
Steroidal drug is that in pharmaceutical industries, output is only second to antibiotic second largest class medicine, steroidal drug has the multiple efficacies such as antianaphylaxis, antishock, anti-inflammatory, anti-allergic, be widely used in the healing for the treatment of bronchial asthma, rheumatic arthritis, eczema, anaemia and promoting fracture and wound, also be used to the endocrinopathys such as treatment Ai Disenshi, have important effect to body development, immunomodulatory, treating skin disease and Birth control aspect.The treatment of steroidal drug to hormone-dependent type tumour has certain curative effect.Along with developing rapidly of steroid chemical, steroid hormone class medicine oneself become the important class of field of medicaments, application is clinically widely.
At present, transform the novel bacterial of steroid drugs about microbial method and new industrial research developing state good.9 α-OH-ADD and 9 α-OH-ADD, as the important source material of synthesizing steroid class medicine and key intermediate, are subject to the favor of more investigator.
Domestic and international research work mainly concentrates in association area and utilizes plant sterol to prepare androstane-4-alkene-3,17-diketone (4-AD) or androstane-1,4-diene-3,17-diketone (ADD), and use the bacterial classification such as Nocardia bacteria or rhodococcus to carry out C to 4-AD or ADD
9 αhydroxylation prepares 9 Alpha-hydroxies-4-AD (9 α-OH-AD) or 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione (9 α-OH-ADD).
(1) sterol prepares 4-AD or ADD
Microorganism for sterol side chain degraded mainly contains mycobacterium (Mycobacterium), Nocardia bacteria (Nocardia), Arthrobacter (Arthrobacterium), tyrothricin (Brevibacterium) etc.Show in numerous document and patent, carry out utilizing plant sterol all to generate 4-AD and ADD in Side chain cleavage process, both generation mass ratioes have different according to the difference of bacterial classification and production technique simultaneously.As adopted bacillus amyloliquefaciens to carry out side chain cleavage to plant sterol in patent CN104403974A, the mass ratio that 4-AD and ADD generates is 18:1; Utilize Mycobacteriumsp.NRRLB3683 to carry out side chain excision to cholesterol in patent CN1544618A, 4-AD and the ADD mass ratio of generation is 1:10.Transformation time is generally all at 100-120h.
(2) 4-AD or ADD prepares 9 α-OH-AD or 9 α-OH-ADD
Microorganism for the reaction of steroidal 9 'alpha '-hydroxylation mainly contains rhodococcus (Rhodococcus), bar bacterium (Corynebacterium), Nocardia bacteria (Nocardia) etc.As patent CN103667126A describes a kind of method utilizing M. smegmatics to prepare 9 α-OH-AD; A kind of method utilizing mycobacterium fortutitum to prepare 9 Alpha-hydroxy Androstenedione is disclosed in patent CN103343155A.Transformation time is generally at about 96-100h.
In sum, just current document and patent, utilized phytosterol bio legal system for 9 α-OH-ADD, need to have been come (i.e. two-step approach) by two-step fermentation, have no the bibliographical information utilizing plant sterol single stage method to prepare 9 α-OH-ADD at present.Two-step approach generally first utilizes mycobacterium that plant sterol is converted into ADD, then through extracting, refining obtain ADD, recycle the bacterial strain such as Nocardia bacteria or rhodococcus afterwards and 9 'alpha '-hydroxylations are carried out to ADD obtain 9 α-OH-ADD.But above-mentioned two-step approach exists following not enough: when 1. plant sterol transforms to ADD direction, the utilising efficiency of plant sterol is on the low side.2. the product that Side chain cleavage fermenting process generates contains 4-AD and ADD all simultaneously, because the structure of 4-AD and ADD is close, is therefore difficult to carry out separation and purification.3. product A DD later stage extraction purification needs to utilize a large amount of suitable organic solvents to carry out Hydrolysis kinetics, to obtain the higher ADD of purity, and could as the substrate of 9 follow-up 'alpha '-hydroxylations.So the industrial biological legal system developing high efficiency and time conservation more has very high using value and prospect for the method for 9 α-OH-ADD.
Summary of the invention
For above-mentioned technical problem of the prior art, the invention provides one and prepare 9 Alpha-hydroxies-androstane-1,4-diene-3, the method of 17-diketone, the method of described this preparation 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione solves in prior art and adopts two-step fermentation to prepare 9 Alpha-hydroxies-androstane-1, the method low conversion rate of 4-diene-3,17-diketone, the technical problem of separation and purification difficulty.
The invention provides one and prepare 9 Alpha-hydroxies-androstane-1,4-diene-3, the method of 17-diketone, take plant sterol as substrate, mycobacterium Mycobacterium and rhodococcus rhodochrous Rhodococcusrhodochrous two kinds of microorganism mixed fungus fermentations are utilized to prepare 9 Alpha-hydroxies-androstane-1,4-diene-3,17-diketone.
Further, described method comprises the steps:
1) step of the fermention medium of a preparation containing plant sterol;
2) in described fermention medium, compound chaotropic agent is added, described compound chaotropic agent is made up of ethanol and polyoxyethylene glycol, in described fermention medium, the concentration of described ethanol is 10.0-50.0g/L, and the concentration of described polyoxyethylene glycol is 5.0-20.0g/L;
3) step of mycobacterium Mycobacterium and rhodococcus rhodochrous Rhodococcusrhodochrous is inoculated respectively for one, the inoculum size of described mycobacterium Mycobacterium is 10.0-15.0%, transform after cultivating 0-48h, access 10.0-20.0% rhodococcus rhodochrous Rhodococcusrhodochrous again, leavening temperature is 28-32 DEG C, fermentation time is 72-120h, and fermention medium pH is between 6.5-7.5;
4) step of a product Hydrolysis kinetics, after fermentation ends, by fermented liquid 70-90 DEG C deactivation, after being cooled to 20-30 DEG C, filter press, gets filter cake and extracts, more after filtration, decolouring, distillation, refining, obtain 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione.Further, the formula of described fermention medium is as follows: glucose 10.0-30.0g/L, primary ammonium phosphate 2.0-4.0g/L, ferric ammonium citrate 0.03-0.08g/L, magnesium sulfate heptahydrate 1.5-3.5g/L, dipotassium hydrogen phosphate 0.5-1.5g/L, potassium primary phosphate 0.5-1.5g/L, beet sirup 5.0-9.0g/L, plant sterol 30.0-40.0g/L.
Further, the preserving number of described mycobacterium Mycobacterium is CICC21097, from Chinese industrial Microbiological Culture Collection administrative center; The preserving number of described rhodococcus rhodochrous Rhodococcusrhodochrous is CGMCC4.1480, from China General Microbiological culture presevation administrative center.
The present invention take plant sterol as substrate, mycobacterium Mycobacterium (CICC21097) and rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480) two kinds of microorganism mixed fungus fermentations are utilized to prepare 9 α-OH-ADD, also by adding compound chaotropic agent in the fermentation medium, bio-transformation is carried out to plant sterol, makes plant sterol Side chain cleavage and C
9 αposition hydroxylation one step completes, and makes plant sterol reach 84.3% to the highest molar yield of 9 α-OH-ADD.
Method of the present invention and two-step approach (namely first make phytosterol bio-conversion be ADD, then are substrate with ADD, at its C
9 αposition hydroxylation obtains 9 α-OH-ADD) to compare and have the following advantages: (1) single stage method is mixed bacterium transformation phytosterin and is prepared 9 α-OH-ADD, its hydroxylation procedures advantageously transforms in plant sterol to ADD direction, improves the utilising efficiency of plant sterol.(2) reduce purification step, eliminate the extraction purification process of ADD in two-step approach, two steps are purified and becomes a step, reduce solvent usage quantity.(3) reduce the generation of by product 4-AD and 9 α-OH-AD, improve the biological transformation ratio of plant sterol to 9 α-OH-ADD, shorten the biological fermentation cycle.
The present invention utilizes synergistic advantage between two bacterial strains, plant sterol is impelled more to transform to 9 α-OH-ADD, reduce the generation of by product 4-AD and 9 α-OH-AD, improve the biologicak efficiency of plant sterol to 9 α-OH-ADD, shorten the biological fermentation cycle, reduce the effect of production cost largely.
The present invention compares with prior art, and its technical progress is significant.The present invention adopts one-step conversion, decreases purification step, namely eliminates the extraction purification process of ADD in two-step approach, two steps is purified and becomes a step, greatly reduce solvent usage quantity, reduce production cost.
Accompanying drawing explanation
Fig. 1 shows the process that mixed bacterium bioconversion phytosterol prepares 9 α-OH-ADD.
Embodiment
Below by specific embodiment, the present invention is set forth further, but do not limit the present invention.
The mycobacterium Mycobacterium (CICC21097) that the present invention adopts is the microorganism of public offering, from, Chinese industrial Microbiological Culture Collection administrative center, the address of this mechanism is: No. 24, Road, Jiuxianqiao, Chaoyang District, Beijing City institute No. 6 building (100015), can be bought by phone, also can pass through ordering site (http://www.china-cicc.org), all can buy as long as anyone pays relevant expense.
The rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480) that the present invention adopts is the microorganism of public offering, from, China General Microbiological culture presevation administrative center, the address of this mechanism is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, can be bought by phone, also can pass through ordering site (http://www.cgmcc.net), all can buy as long as anyone pays relevant expense.
Embodiment 1
Mycobacterium Mycobacterium (CICC21097) and rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480), by slant culture and seed culture, obtains the bacterial classification required for fermenting.One, mycobacterium Mycobacterium (CICC21097) slant strains and seed liquor preparation
(1) slant culture
Slant medium: peptone 5.0g/L, extractum carnis 3.0g/L, sodium-chlor 5.0g/L, glucose 10.0g/L, agar 20.0g/L, cultivate 5-7 days under 29 DEG C of conditions.
(2) seed culture
Seed culture medium: glucose 15.0g/L, peptone 5.0g/L, extractum carnis 3.0g/L, sodium-chlor 5.0g/L, magnesium sulfate heptahydrate 2.5g/L, primary ammonium phosphate 3.5g/L, dipotassium hydrogen phosphate 1.0g/L, potassium primary phosphate 1.0g/L, pH7.0,121 DEG C of moist heat sterilization 30min.
By the bacterial strain that step (1) is cultivated, aseptically inoculate 2 rings in the shaking flask of 250mL that 50mL seed culture medium is housed with transfering loop, under 30 DEG C of conditions, rotary shaker 200r/min cultivates 48h, obtained seed liquor.
Two, rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480) slant strains and seed liquor preparation
(1) slant culture
Slant medium: glucose 10.0g/L, Tryptones 15.0g/L, soy peptone 5.0g/L, sodium-chlor 5.0g/L, agar 20.0g/L, cultivate 5-6 days under 30 DEG C of conditions.
(2) seed culture
Seed culture medium: glucose 10.0g/L, yeast extract paste 20.0g/L, fish peptone 5.0g/L, W-Gum 9.0g/L, dipotassium hydrogen phosphate 2.5g/L, pH7.0,121 DEG C of moist heat sterilization 30min.
By the bacterial strain that step (1) is cultivated, aseptically inoculate 1 ring in the shaking flask of 250mL that 50mL seed culture medium is housed with transfering loop, under 30 DEG C of conditions, rotary shaker 200r/min cultivates 30h, obtained seed liquor.
Three, fermentation culture
Fermention medium: plant sterol 35.0g/L, glucose 21.0g/L, primary ammonium phosphate 3.5g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate heptahydrate 2.0g/L, dipotassium hydrogen phosphate 1.2g/L, potassium primary phosphate 1.0g/L, beet sirup 6.0g/L, pH7.0.
The fermentor tank of fermention medium will be housed in 121 DEG C of high pressure steam sterilization 15min.
Cultivate and initially add compound chaotropic agent in the medium: ethanol 30.0g/L, polyoxyethylene glycol 10.0g/L.Inoculate again, the inoculum size of mycobacterium MycobacteriumCICC (21097) is 12.0%, at the Simultaneous vaccination rhodococcus rhodochrous of mycobacterium inoculation, the inoculum size of rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480) is 17.0%, and use 5L fermentor tank to carry out bio-transformation, liquid amount is 70%, air flow is 1.0v/vmin, temperature controls at 30 DEG C, and rotating speed is 300r/min, samples TLC monitoring between transition phase every 12h.Fermentation culture 96h, sampling TLC analyzes, and without plant sterol spot, transforms and substantially completes.Sample is extracted with ethyl acetate, and collected by centrifugation supernatant liquor adopts the content of 9 α-OH-ADD in the fermented liquid of the above-mentioned gained of high-efficient liquid phase chromatogram technique analysis to be 17.5g/L, and the content of 9 α-OH-AD is 0.25g/L, and plant sterol molar yield is 72.4%.
After fermentation ends, by fermented liquid 85 DEG C of deactivations, after being cooled to 25 DEG C, filter press, filter cake dichloromethane extraction, more after filtration, decolouring and the operation such as distillation obtain 9 α-OH-ADD crude products, crude product is refining with toluene making beating again, finally obtains 9 α-OH-ADD fine work.High-efficient liquid phase chromatogram technique analysis purity is 98.7%.
Embodiment 2
Mycobacterium Mycobacterium (CICC21097) and rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480) bacterial strain are made seed liquor (method is with embodiment 1), then carries out fermentation culture.
Fermention medium: plant sterol 35.0g/L, ethanol 31.0g/L, polyoxyethylene glycol 9.0g/L, glucose 20.0g/L, primary ammonium phosphate 3.0g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate heptahydrate 2.5g/L, dipotassium hydrogen phosphate 1.0g/L, potassium primary phosphate 1.0g/L, beet sirup 6.0g/L, pH7.0.
The fermentor tank of fermention medium will be housed in 121 DEG C of high pressure steam sterilization 15min.
Cultivate and initially add compound chaotropic agent in the medium: ethanol 31.0g/L, polyoxyethylene glycol 9.0g/L.Inoculate again, the inoculum size of mycobacterium MycobacteriumCICC (21097) is 12.0%, rhodococcus rhodochrous is inoculated after mycobacterium transforms cultivation 24h, the inoculum size of rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480) is 18.0%, and use 5L fermentor tank to carry out bio-transformation, liquid amount is 70%, air flow is 1.0v/vmin, temperature controls at 30 DEG C, and rotating speed is 300r/min, samples TLC monitoring between transition phase every 12h.Fermentation culture 96h, sampling TLC analyzes, and without plant sterol spot, transforms and substantially completes.Sample is extracted with ethyl acetate, and collected by centrifugation supernatant liquor adopts the content of 9 α-OH-ADD in the fermented liquid of the above-mentioned gained of high-efficient liquid phase chromatogram technique analysis to be 20.4g/L, and the content of 9 α-OH-AD is 0.19g/L, and plant sterol molar yield is 84.3%.
After fermentation ends, by fermented liquid 85 DEG C of deactivations, after being cooled to 25 DEG C, filter press, filter cake dichloromethane extraction, more after filtration, decolouring and the operation such as distillation obtain 9 α-OH-ADD crude products, crude product is refining with toluene making beating again, finally obtain 9 α-OH-ADD fine work, high-efficient liquid phase chromatogram technique analysis purity is 99.1%.
Embodiment 3
Mycobacterium Mycobacterium (CICC21097) and rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480) bacterial strain make seed liquor (method is with embodiment 1), then carry out fermentation culture.
Fermention medium: plant sterol 35.0g/L, glucose 22.0g/L, primary ammonium phosphate 3.0g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate heptahydrate 2.0g/L, dipotassium hydrogen phosphate 1.0g/L, potassium primary phosphate 1.0g/L, beet sirup 6.0g/L, pH7.0.
The fermentor tank of fermention medium will be housed in 121 DEG C of high pressure steam sterilization 15min.
Cultivate and initially add compound chaotropic agent in the medium: ethanol 30.0g/L, polyoxyethylene glycol 9.0g/L.Inoculate again, the inoculum size of mycobacterium MycobacteriumCICC (21097) is 13.0%, rhodococcus rhodochrous is inoculated after mycobacterium transforms cultivation 48h, the inoculum size of rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480) is 17.0%, and use 5L fermentor tank to carry out bio-transformation, liquid amount is 70%, air flow is 1.0v/vmin, temperature controls at 30 DEG C, and rotating speed is 300r/min, samples TLC monitoring between transition phase every 12h.Fermentation culture 96h, sampling TLC analyzes, and without plant sterol spot, transforms and substantially completes.Sample is extracted with ethyl acetate, and collected by centrifugation supernatant liquor adopts the content of 9 α-OH-ADD in the fermented liquid of the above-mentioned gained of high-efficient liquid phase chromatogram technique analysis to be 18.3g/L, and the content of 9 α-OH-AD is 0.31g/L, and plant sterol molar yield is 75.7%.
After fermentation ends, by fermented liquid 85 DEG C of deactivations, after being cooled to 25 DEG C, filter press, filter cake dichloromethane extraction, more after filtration, decolouring and the operation such as distillation obtain 9 α-OH-ADD crude products, crude product is refining with toluene making beating again, finally obtain 9 α-OH-ADD fine work, high-efficient liquid phase chromatogram technique analysis purity is 98.6%.
Embodiment 4
Mycobacterium Mycobacterium (CICC21097) and rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480) bacterial strain make seed liquor (method is with embodiment 1), then carry out fermentation culture.
Fermention medium: plant sterol 30.0g/L, glucose 10.0g/L, primary ammonium phosphate 2.0g/L, ferric ammonium citrate 0.08g/L, magnesium sulfate heptahydrate 3.5g/L, dipotassium hydrogen phosphate 1.5g/L, potassium primary phosphate 1.5g/L, beet sirup 9.0g/L, pH7.5.
The fermentor tank of fermention medium will be housed in 121 DEG C of high pressure steam sterilization 15min.
Cultivate and initially add compound chaotropic agent in the medium: ethanol 10.0g/L, polyoxyethylene glycol 5.0g/L.Inoculate again, the inoculum size of mycobacterium MycobacteriumCICC (21097) is 10.0%, rhodococcus rhodochrous is inoculated after mycobacterium transforms cultivation 24h, the inoculum size of rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480) is 10.0%, and use 5L fermentor tank to carry out bio-transformation, liquid amount is 70%, air flow is 1.0v/vmin, temperature controls at 28 DEG C, and rotating speed is 300r/min, samples TLC monitoring between transition phase every 12h.Fermentation culture 72h, sampling TLC analyzes, and without plant sterol spot, transforms and substantially completes.Sample is extracted with ethyl acetate, and collected by centrifugation supernatant liquor adopts the content of 9 α-OH-ADD in the fermented liquid of the above-mentioned gained of high-efficient liquid phase chromatogram technique analysis to be 17.1g/L, and the content of 9 α-OH-AD is 0.20g/L, and plant sterol molar yield is 82.5%.
After fermentation ends, by fermented liquid 85 DEG C of deactivations, after being cooled to 25 DEG C, filter press, filter cake dichloromethane extraction, more after filtration, decolouring and the operation such as distillation obtain 9 α-OH-ADD crude products, crude product is refining with toluene making beating again, finally obtain 9 α-OH-ADD fine work, high-efficient liquid phase chromatogram technique analysis purity is 98.8%.
Embodiment 5
Mycobacterium Mycobacterium (CICC21097) and rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480) bacterial strain make seed liquor (method is with embodiment 1), then carry out fermentation culture.
Fermention medium: plant sterol 40.0g/L, glucose 30.0g/L, primary ammonium phosphate 4.0g/L, ferric ammonium citrate 0.03g/L, magnesium sulfate heptahydrate 1.5g/L, dipotassium hydrogen phosphate 0.5g/L, potassium primary phosphate 0.5g/L, beet sirup 5.0g/L, pH6.5.
The fermentor tank of fermention medium will be housed in 121 DEG C of high pressure steam sterilization 15min.
Cultivate and initially add compound chaotropic agent in the medium: ethanol 50.0g/L, polyoxyethylene glycol 20.0g/L.Inoculate again, the inoculum size of mycobacterium MycobacteriumCICC (21097) is 15.0%, rhodococcus rhodochrous is inoculated after mycobacterium transforms cultivation 24h, the inoculum size of rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480) is 20.0%, and use 5L fermentor tank to carry out bio-transformation, liquid amount is 70%, air flow is 1.0v/vmin, temperature controls at 32 DEG C, and rotating speed is 300r/min, samples TLC monitoring between transition phase every 12h.Fermentation culture 120h, sampling TLC analyzes, and without plant sterol spot, transforms and substantially completes.Sample is extracted with ethyl acetate, and collected by centrifugation supernatant liquor adopts the content of 9 α-OH-ADD in the fermented liquid of the above-mentioned gained of high-efficient liquid phase chromatogram technique analysis to be 21.5g/L, and the content of 9 α-OH-AD is 0.24g/L, and plant sterol molar yield is 77.8%.
After fermentation ends, by fermented liquid 85 DEG C of deactivations, after being cooled to 25 DEG C, filter press, filter cake dichloromethane extraction, more after filtration, decolouring and the operation such as distillation obtain 9 α-OH-ADD crude products, crude product is refining with toluene making beating again, finally obtain 9 α-OH-ADD fine work, high-efficient liquid phase chromatogram technique analysis purity is 98.1%.
Comparative example 1
Mycobacterium Mycobacterium (CICC21097) bacterial strain makes seed liquor (method is with embodiment 1), then carries out fermentation culture.
Fermention medium as described in Example 2, will be equipped with the fermentor tank of fermention medium in 121 DEG C of high pressure steam sterilization 15min.
Cultivate and initially add compound chaotropic agent in the medium: ethanol 31.0g/L, polyoxyethylene glycol 9.0g/L.Inoculate again, the inoculum size of mycobacterium Mycobacterium (CICC21097) is 12.0%, 5L fermentor tank is used to carry out bio-transformation, liquid amount is 70%, air flow is 1.0v/vmin, temperature controls at 30 DEG C, and rotating speed is 300r/min, samples TLC monitoring between transition phase every 12h.Fermentation culture 96h, sampling TLC analyzes, and transforms and stagnates.Sample is extracted with ethyl acetate, and in the fermented liquid of the above-mentioned gained of collected by centrifugation supernatant liquor employing high-efficient liquid phase chromatogram technique analysis, the content of ADD is the content of 16.7g/L, 4-AD is 0.87g/L, and plant sterol molar yield is 72.9%.
After fermentation ends, by fermented liquid 85 DEG C of deactivations, after being cooled to 25 DEG C, filter press, filter cake dichloromethane extraction, more after filtration, decolouring and the operation such as distillation obtain ADD crude product, crude product is refining with toluene making beating again, and finally obtain ADD fine work, high-efficient liquid phase chromatogram technique analysis purity is 98.5%.
Comparative example 2
The bacterial strain of rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480) makes seed liquor (method is with embodiment 1), then carries out fermentation culture.
As described in Example 2, substrate changes the ADD of 24.0g/L into by the plant sterol of 35.0g/L to fermention medium.The fermentor tank of fermention medium will be housed in 121 DEG C of high pressure steam sterilization 15min.
Cultivate and initially add compound chaotropic agent in the medium: ethanol 31.0g/L, polyoxyethylene glycol 9.0g/L.Inoculate again, the inoculum size of rhodococcus rhodochrous Rhodococcusrhodochrous (CGMCC4.1480) is 18.0%, 5L fermentor tank is used to carry out bio-transformation, liquid amount is 70%, air flow is 1.0v/vmin, temperature controls at 30 DEG C, and rotating speed is 300r/min, samples TLC monitoring between transition phase every 12h.Fermentation culture 72h, sampling TLC analyzes, and without ADD spot, transforms and substantially completes.Sample is extracted with ethyl acetate, and it is 93.8% that collected by centrifugation supernatant liquor adopts the content of 9 α-OH-ADD in the fermented liquid of the above-mentioned gained of high-efficient liquid phase chromatogram technique analysis to be 22.6g/L, ADD molar yield.
After fermentation ends, by fermented liquid 85 DEG C of deactivations, after being cooled to 25 DEG C, filter press, filter cake dichloromethane extraction, more after filtration, decolouring and the operation such as distillation obtain 9 α-OH-ADD crude products, crude product is refining with toluene making beating again, finally obtain 9 α-OH-ADD fine work, high-efficient liquid phase chromatogram technique analysis purity is 99.1%.
Known by embodiment, single stage method is compared with two-step approach, and single stage method has following advantage:
1. the transformation efficiency improving plant sterol is contributed to.As described in comparative example 1 and 2, two-step approach bioconversion phytosterol prepares 9 α-OH-ADD, and the whole molar yield of plant sterol is 72.9% × 93.8%=68.4%; And as described in Example 2, single stage method bioconversion phytosterol prepares 9 α-OH-ADD, plant sterol molar yield is up to 84.3%.
2. contribute to shortening fermentation period.As described in comparative example 1 and 2, the two-step approach microbial transformation time is about 168h, and as described in Example 2, single stage method mixed fermentation is produced 9 α-OH-ADD bioconversion time and is about 96h, and the bioconversion time of single stage method shortens about 72h than two-step approach.
3. solvent usage quantity is reduced.Single stage method eliminates the extraction purification process of ADD in two-step approach, two steps is purified and becomes a step, reduce solvent load, reduce production cost.
4. the generation of Main By product 4-AD and 9 α-OH-AD is reduced.Can be found out with comparing of comparative example 1 by embodiment 2, when utilizing mycobacterium to carry out single bacterium bio-transformation to plant sterol, have the generation of ADD and 4-AD, and both mass ratioes be about 19:1 simultaneously.And when utilizing two kinds of bacterium mixed fermentations to produce 9 α-OH-ADD, have the generation of 9 α-OH-ADD and 9 α-OH-AD, and both mass ratioes are about 107:1 simultaneously.Namely single stage method is mixed bacterium transformation phytosterin and is produced the generation that 9 α-OH-ADD effectively can reduce by product 4-AD and 9 α-OH-AD, and its hydroxylation procedures advantageously transforms in plant sterol to ADD direction, improves the utilising efficiency of plant sterol.
Foregoing be only the present invention conceive under basic explanation, and according to any equivalent transformation that technical scheme of the present invention is done, all should protection scope of the present invention be belonged to.
Claims (4)
1. prepare a method for 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione, it is characterized in that: be substrate with plant sterol, utilize mycobacterium
mycobacteriumand rhodococcus rhodochrous
rhodococcusrhodochroustwo kinds of microorganism mixed fungus fermentations prepare 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione.
2. a kind of method preparing 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione according to claim 1, is characterized in that: comprise the steps:
1) step of the fermention medium of a preparation containing plant sterol;
2) in described fermention medium, compound chaotropic agent is added, described compound chaotropic agent is made up of ethanol and polyoxyethylene glycol, in described fermention medium, the concentration of described ethanol is 10.0-50.0g/L, and the concentration of described polyoxyethylene glycol is 5.0-20.0g/L;
3) mycobacterium is inoculated respectively for one
mycobacteriumand rhodococcus rhodochrous
rhodococcusrhodochrousstep, mycobacterium
mycobacteriuminoculum size be 10.0-15.0%, transform and cultivate after 0-48h, then access the rhodococcus rhodochrous of 10.0-20.0%
rhodococcusrhodochrous, leavening temperature is 28-32 DEG C, and fermentation time is 72-120h, and fermention medium pH is between 6.5-7.5;
4) step of a product Hydrolysis kinetics, after fermentation ends, by fermented liquid 70-90 DEG C deactivation, after being cooled to 20-30 DEG C, filter press, gets filter cake and extracts, more after filtration, decolouring, distillation, refining, obtain 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione.
3. one according to claim 1 prepares 9 Alpha-hydroxies-androstane-1,4-diene-3, the method of 17-diketone, is characterized in that the formula of described fermention medium is as follows: glucose 10.0-30.0g/L, primary ammonium phosphate 2.0-4.0g/L, ferric ammonium citrate 0.03-0.08g/L, magnesium sulfate heptahydrate 1.5-3.5g/L, dipotassium hydrogen phosphate 0.5-1.5g/L, potassium primary phosphate 0.5-1.5g/L, beet sirup 5.0-9.0g/L, plant sterol 30.0-40.0g/L.
4. a kind of method preparing 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione according to claim 1, is characterized in that: described mycobacterium
mycobacteriumpreserving number be CICC21097, from Chinese industrial Microbiological Culture Collection administrative center; Described rhodococcus rhodochrous
rhodococcusrhodochrouspreserving number be CGMCC4.1480, from China General Microbiological culture presevation administrative center.
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