CN109852658A - A method of boldenone is prepared using microorganism conversion - Google Patents
A method of boldenone is prepared using microorganism conversion Download PDFInfo
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Abstract
The invention discloses a kind of methods for preparing boldenone, convert boldenone for Isosorbide-5-Nitrae-androstenedione (ADD) using saccharomycete, the saccharomycete is saccharomyces cerevisiaeSaccharomyces cerevisiaeCGMCC 2.346 or saccharomyces uvarumSaccharomyces uvarum One of CGMCC 3.4636.The beneficial effect that the present invention reaches is: 1, strong reductant poor selectivity, process in chemical method are effectively prevented using bioconversion can not meet the defect of production process Environmental Safety requirement；2, culture medium is cheap and easy to get, and culture conversion temperature is higher, growth rapidly, it is not stringent to sterile requirement the advantages that；3, present invention process prepares that boldenone reaction route is short, and product quality is excellent, is suitble to industrialized production.
Description
Technical field
The invention belongs to steroidal field of biotechnology, in particular to a kind of side that boldenone is prepared using microorganism conversion
Method.
Background technique
Boldenone (17 beta-hydroxies-Isosorbide-5-Nitrae-androstane diene -3- ketone, CAS:846-48-0), also known as boldenone, are protein assimilatings
One kind of agent has the effects that enhance blood vessel oxygen delivery capacity, enhances muscle, highlight blood vessel, increase appetite.Structural formula are as follows:
About the preparation method of boldenone, domestic enterprise is mostly using chemical synthesis process, from 4-AD at present
(AD) it is prepared with Isosorbide-5-Nitrae-androstenedione (ADD).
Route one: preparing boldenone from 4-AD (AD) starting, need with the reducing agents of potassium boron hydrogen species to 17 into
Row reduction prepares testosterone (TS), then recycles microorganism or chemical method to carry out 1,2 dehydrogenations and boldenone is made.This method
One step restores the reducing agent for needing to use potassium boron hydrogen species, and reduction reaction can simultaneously carry out 3 and 17 ketone groups, and impurity is more,
Yield is not high.For the side reaction for avoiding 3 ketone groups, it need to generally increase ketone group protection and deprotection steps, and dehydrogenation step then needs
Bacterium dehydrogenation is used, or makees dehydrating agent using the more expensive DDQ of price.That there is only routes is longer, ropy for this method
Problem, and in dehydrogenation stage, it is very strict to sterility requirements in culture and conversion process using bacterium dehydrogenation, use DDQ
Then product quality is poor for dehydrogenation.
Route two: boldenone is prepared from Isosorbide-5-Nitrae-androstenedione (ADD).Patent CN103030677 report, first to Isosorbide-5-Nitrae-hero
Alkene diketone (ADD) carries out etherification protection, then restores 17 ketone groups, obtains boldenone after deprotection；Patent
CN104327143 then improves the selectivity of reducing agent by optimization reaction condition and obtains boldenone come a step.Though both methods
So it is greatly improved compared with preparing boldenone route from 4-AD (AD) starting, but all unavoidable high reproducibility chemistry examination
The use of agent, there are very high security risks in production, storage and transportation, use for these reducing agents, the production requirement with safe green
It runs counter to.
And the selective reduction enzyme of 17 ketone groups on steroidal is prevalent in multiple-microorganism, it filters out suitable micro-
Biology simultaneously cooperates zymotechnique appropriate to complete step reaction, and scientific worker is in unremitting effort.Due to the fermentoid be belong to compared with
For complicated oxidoreducing enzyme, and enzyme activity is not high in most microorganisms, is not enough to be formed competitive commercialization process.Such as
Inventor finds that during bacterial screening, candida utili Candida utilis AS2.1180 and
AS2.1461 does not have activity of conversion, Candida parapsilosis Candida parapsilosis GIM2.190 and GIM2.146
There is activity of conversion, but conversion capability is lower, is not enough to be formed competitive commercialization process, Arthrobacter simplex
Arthrobacter simplex CPCC 140451 and rhodococcus erythropolis Rhodococcus erythropolis ATCC
17896 have activity of conversion, but conversion capability is lower and generates more other impurities.In addition, the enzyme is usually intracorporal by microorganism
Nutritional status regulation can strengthen back reaction, the hydroxyl of reduction is oxidized to ketone group again when nutrition is poor, so that final turns
Rate is lower, is unfavorable for isolating and purifying.In Chinese patent CN102168099, inventor is even by 1,2 dehydrogenase bases of steroidal
Because being transferred in Pichia pastoris, the intracorporal dehydrogenation to 4-AD (AD)-keto reductase catalysis route is constructed, but throw
Material concentration only has 0.15%, and commercialization value is little.
Therefore, suitable microorganism is searched out, and it is environmentally protective, healthy and safe to establish production process, product yield, quality
With cost there is the conversion process of commercialization competitiveness to have great importance.
Summary of the invention
The purpose of the present invention is: for the defect of prior art preparation boldenone method, it is high to find a kind of selectivity, and pacifies
Entirely, the environmentally protective method for preparing boldenone.Inventor screens two plants of saccharomyces bacterium from more plants of microbial strains,
17 one steps of ketone group of Isosorbide-5-Nitrae-androstenedione are selectively reduced to β hydroxyl, obtain boldenone using purification procedures,
Isosorbide-5-Nitrae-androstenedione feed concentrations are 0.5-0.75%；Microorganism fungus kind described here refers to saccharomyces cerevisiae
Saccharomyces cerevisiae (China General Microbiological Culture Collection Center CGMCC 2.346) or saccharomyces uvarum
Saccharomyces uvarum (China General Microbiological Culture Collection Center CGMCC 2.4636).
The specific transformation routes of the present invention are as follows:
The microorganism used: the Saccharomyces Cerevisiae in S accharomyces cerevisiae CGMCC that the present invention uses
2.346, China General Microbiological Culture Collection Center is purchased from July, 1996, the strain original preservation time is January nineteen fifty-two
1, primary source was DaLian, China Institute of Microorganism, Academia Sinica, and original deposit number is Dalian Y355；The present invention makes
Saccharomyces uvarum Saccharomyces uvarum CGMCC 2.4636 is purchased from China General Microbiological in June, 2015
Culture Collection Center, the strain is initially from Portugal Nahuel Huapi National Park, Northwestern
Patuyonia is collected, after by the white seam man of virtue and ability of preservation people on November 13rd, 2012 in China General Microbiological culture presevation
Heart preservation, deposit number are CGMCC 2.4636.
Specifically, scheme provided by the invention includes the following steps:
(1) culture medium is prepared
Prepare seed culture medium, conversion culture medium and feed supplement nutrient solution.Wet-hot steam sterilizing is carried out, it is standby to be cooled to room temperature
With.
Glucose 4.5% in above-mentioned seed culture medium, corn pulp 2.0%, adjusting pH value are 4- 4.5.
Carbon source concentration is 2-6.5% in above-mentioned conversion culture medium, may is that glucose, maltose, sucrose, dextrin, glycerol
One of, further preferably glucose；
Nitrogen concentration is 1-3% in above-mentioned conversion culture medium, comprising: yeast extract, yeast powder, peptone, corn pulp, urine
Element, ammonium salt etc., further preferably corn pulp；
The inorganic salts ingredients such as phosphate, sylvite and sodium salt can be added in above-mentioned conversion culture medium in right amount.
In above-mentioned feed supplement nutrient solution, carbon source nutrient solution is glucose solution, and nitrogen source nutrient solution is yeast extract aqueous solution.
(2) seed culture
Strain is accessed under aseptic condition in seed culture medium, after cultivation temperature and shaking speed is arranged, culture 16-30 is small
When；
Above-mentioned strain is Saccharomyces Cerevisiae in S accharomyces cerevisiae (China General Microbiological Culture Collection Center
CGMCC 2.346) or saccharomyces uvarum Saccharomyces uvarum (China General Microbiological Culture Collection Center CGMCC
One of 2.4636).
Above-mentioned 28-35 DEG C of cultivation temperature section, further preferably 30 DEG C；
Above-mentioned condition of culture includes shaking speed, is 160-200 rpms.
(3) Spawn incubation is converted
After seed culture, appropriate seed liquor is accessed into conversion culture medium, culture transferring amount is 5%- 20%, in culture temperature
Degree is cultivated 14-24 hours between 28-35 DEG C and under the conditions of shaking speed is 160-200 rpms.
Above-mentioned culture transferring amount be 5%-20%, most preferably 20%.
Above-mentioned 28-35 DEG C of cultivation temperature section, further preferably 30 DEG C.
(4) feed intake conversion, period feed supplement is built up one's health
After conversion Spawn incubation is good, directly feeds intake or construct conversion of resting cells system and feed intake, later under proper condition
Conversion.Period feed supplement is built up one's health conversion.
Building conversion of resting cells system, which refers to, among the above collects yeast thallus for the method for converting strain centrifugation, uses phosphorus
After acid buffer washing, then it is resuspended in and is converted in shaking flask with the fermentation isometric phosphate buffer of centrifugate.
Above-mentioned phosphate buffer is advisable with the 0.1M PBS buffer solution of pH6.0-7.0.
It feeds intake and can be the Isosorbide-5-Nitrae-androstenedione (ADD) for direct plungeing into micronization, it is desirable that sieve with 100 mesh sieve net；Substrate can also
Being put into after ethyl alcohol or isopropanol dissolved clarification；Primary or gradation is put into, total amount of feeding 0.5%-0.75%, ethyl alcohol or isopropyl
The dosage of alcohol is the 1-3 times of volume (1-3V) of ADD.
Above-mentioned feed supplement interval 8-14 hours, 0.25% micronization of investment or with Isosorbide-5-Nitrae-androstenedione after solvent dissolved clarification every time
(ADD), amounting to inventory is 0.5%-0.75%.
Nutritional supplementation liquid should be also filled into while supplementing substrate, direct fermentation transform mode fills into glucose 1%, ferment every time
Female cream 0.3%；Resting cell system only supplements glucose.It builds up one's health to be no more than 5 times and be advisable, supplement interval is also small for 8-14
When.
(5) heating terminates fermentation, separation and purification.
After microbe conversion, strain is inactivated by heating, the blending filter cake of thallus and material is harvested with the method for filtering,
It is extracted with dissolutions such as acetone, methanol and obtains conversion crude product, acquisition fine work is refined by ethyl acetate and hexamethylene mixing dissolved,
Mother liquor returns throwing microbe conversion.
To improve the transformation efficiency of strain, and raw material is avoided to poison to the inhibiting effect of microbiota metabolic activity, to being thrown
Material is fed intake using micronization or dissolution, with optimization dispersion and mass transport process；Concentration of substrate is avoided by the way of step feeding
The excessively high serious activity of conversion for inhibiting microorganism.
To avoid microorganism during the fermentation, when nutriment is poor, reduced again to material oxidation has been converted
Conversion ratio maintains the metabolism state and conversion capability of microorganism, to improve by way of machine extra-nutrition object in due course
Feed concentrations and conversion ratio.
Opportunity appropriate is selected to stop conversion process, to avoid the generation and reduction adverse effect of unfavorable process.
In order to clearly explain the solution of the present invention, it is not optimised in embodiment at one, uses saccharomyces cerevisiae
Saccharomyces cerevisiae (China General Microbiological Culture Collection Center CGMCC 2.346), using micronization one
It is secondary to feed intake and direct fermentation transform mode, conversion ratio 72.27%；
Preferably, in one embodiment, using Saccharomyces Cerevisiae in S accharomyces cerevisiae (China
General Microbiological Culture collection CGMCC 2.346), it is fed intake and direct fermentation transform mode, is fermented the phase using solvent dissolution
Between interval add substrate and nutrient solution, conversion ratio 92.43%.
Preferably, in one embodiment, using saccharomyces uvarum Saccharomyces uvarum, (China is general
Logical Culture Collection CGMCC 2.4636), it is fed intake and direct fermentation transform mode, is fermented the phase using micronizing materials
Between interval add substrate and nutrient solution, conversion ratio 90.99%.
Preferably, in one embodiment, building conversion of resting cells system mode converts and micronization feeds intake,
Substrate and nutrient solution, conversion ratio 86.98% are added in interval during fermentation.
Compared with prior art, the beneficial effect that the present invention reaches is:
(1) the two plants of strains filtered out using the present invention, Saccharomyces Cerevisiae in S accharomyces cerevisiae or grape
Juice yeast Saccharomyces uvarum restores 17 ketone groups of Isosorbide-5-Nitrae-androstenedione (ADD) to prepare boldenone, and ADD is thrown
Material concentration is 0.5%- 0.75%；After conversion, then with acetone or methanol by extracting, decoloration, concentration are filtered and are dried
It is dry and etc. to obtain conversion crude product；Crude product ethyl acetate and hexamethylene are mixed into dissolved purification, it is final to obtain boldenone fine work.
85% or more ADD conversion ratio, boldenone yield 85%-90%, 99.5% or more purity.It is effectively prevented using bioconversion
Strong reductant poor selectivity, process in chemical method can not meet the defect of production process Environmental Safety requirement.
(2) culture medium that the present invention uses is cheap and easy to get, and culture conversion temperature is higher, and growth is rapid, to sterile requirement
The advantages that not stringent.It is repeatedly fed intake feed supplement, is breached in traditional steroidal zymotechnique because of substrate using high activity strain simultaneously
The limitation low to feed concentrations caused by thallus toxic action improves feed concentrations and conversion ratio, while mother liquor returns throwing, improves
The yield of product has commercial cost advantage.
(3) prepare that boldenone reaction route is short using present invention process, and also using the strong ketone group of specificity in microorganism
Protoenzyme catalysis, fermentation crude product are not in protection, the incomplete residual of deprotection reaction on the reduction impurity and ketone group of 3 ketone groups
The impurity occurred in the chemical synthesis routes such as object, therefore the technical solution provided by the invention for preparing boldenone is economical and practical, instead
Mild condition is answered, obtained product quality is excellent, is suitble to industrialized production.
Detailed description of the invention
Fig. 1 is the HPLC spectrogram of 3 substrate A DD conversion ratio of embodiment；
Fig. 2 is the HPLC spectrogram of 4 substrate A DD conversion ratio of embodiment；
Fig. 3 is the HPLC spectrogram of 5 substrate A DD conversion ratio of embodiment；
Fig. 4 is opposite appearance time of the substrate A DD in HPLC spectrogram.
Specific embodiment
Below with reference to embodiment, the content of the present invention will be explained in more detail.
In the present invention, all equipment and raw material etc. are commercially available or the industry is common.Following implementations
Method in example is unless otherwise instructed the conventional method of this field.
Primary raw material explanation:
The present invention is to prepare boldenone in a manner of Microbial Biotransformation Isosorbide-5-Nitrae-androstenedione (ADD).Make in embodiment
Saccharomycete is Saccharomyces Cerevisiae in S accharomyces cerevisiae (China General Microbiological Culture Collection Center CGMCC
Or saccharomyces uvarum Saccharomyces uvarum (China General Microbiological Culture Collection Center CGMCC 2.346)
2.4636)。
Microorganism conversion is 17 keto reductases using microorganism, and the Land use systems of the enzyme include that growth cell turns
Change, conversion of resting cells.
The investing method of substrate include solid feed intake with solvent dissolution feed intake.
Strain inclined plane culture use yeast extract peptone glucose (YPD) agar medium, 30 DEG C culture 3 days after in 4 DEG C protect
Hiding.
YPD culture medium prescription: 2% agar powder is added in 1% yeast extract, 2% peptone, 2% glucose.
Seed culture medium proportion: glucose 4.5%, corn pulp 2.0%.Sterilising conditions are 115 DEG C, 15 minutes.
Convert Medium Proportion: glucose 5.5%, corn pulp 2.0%, yeast extract 0.9%, potassium dihydrogen phosphate 0.68%,
Sodium hydroxide 0.13%, pH6.2-6.4.Sterilising conditions are 115 DEG C, 15 minutes.
10% glucose is used to supplement as carbon source, 5% yeast extract is supplemented as nitrogen source.Sterilising conditions are 115 DEG C,
10 minutes.
Embodiment 1
Seed liquor shaking flask is prepared, glucose 4.5%, corn pulp 2.0%, adjusting pH value is 4- 4.5.Use 500 milliliter three
Angle bottle, 100 milliliters of liquid amount, 115 DEG C sterilize for 15 minutes, after being cooled to room temperature, an oese saccharomyces cerevisiae are accessed, in 30 DEG C
160 rpms shaking table culture 24 hours；Prepare fermentation liquid shaking flask, glucose 5.5%, corn pulp 2.0%, yeast extract
0.9%, potassium dihydrogen phosphate 0.68%, sodium hydroxide 0.13%, pH6.2-6.4, with 500 milliliters of triangular flasks, 100 milli of liquid amount
It rises, 115 DEG C sterilize for 15 minutes.It is cooled to room temperature.
Every fermentation liquid shaking flask moves into 5 milliliters of strain liquid, in 30 DEG C 160 rpms of shaking table cultures 16 hours, every bottle of investment
0.25 gram of Isosorbide-5-Nitrae-androstenedione of 100 meshes is smashed it through, is continued microbe conversion 24 hours, is inactivated within 30 minutes in 80 DEG C.5 are shaken
Bottle fermentation liquid is filtered with filter paper, and filter cake merges after being extracted 2 times with 10 times of vol acetones, and centrifugal discharge obtains crude product, crude product after concentration
Oven-dried weight 0.97g, with methanol dissolution sample presentation detection, conversion ratio 72.27%.0.8-1.5 times of volume ethyl acetate dissolved clarification of crude product
Afterwards, 5-10 times of volume hexamethylene dilution crystallization is added to refine 3 times, obtains boldenone (BD) fine work 0.46g, HPLC content
99.2%.
Embodiment 2
Seed liquor shaking flask is prepared, glucose 4.5%, corn pulp 2.0%, adjusting pH value is 4- 4.5.Use 500 milliliter three
Angle bottle, 100 milliliters of liquid amount, 115 DEG C sterilize for 15 minutes, after being cooled to room temperature, an oese saccharomyces cerevisiae are accessed, in 30 DEG C
180 rpms shaking table culture 24 hours；Prepare fermentation liquid shaking flask, glucose 5.5%, corn pulp 2.0%, yeast extract
0.9%, potassium dihydrogen phosphate 0.68%, sodium hydroxide 0.13%, pH6.2-6.4, with 500 milliliters of triangular flasks, 100 milli of liquid amount
It rises, 115 DEG C sterilize for 15 minutes.It is cooled to room temperature.
Every fermentation liquid shaking flask moves into 10 milliliters of strain liquid, in 30 DEG C of 180 rpms of shaking table cultures 20 hours investments, 2 times of second
0.25 gram of Isosorbide-5-Nitrae-androstenedione of 2 times of ethyl alcohol dissolution is put into after 0.25 gram of Isosorbide-5-Nitrae-androstenedione, 12 hours of alcohol dissolution again, together
When add nutritive dextrose 1%, it is primary to add nutrition for every 12 hours later for yeast extract 0.3%, until heating up out after 96 hours
Material, 5 shaking flasks, which merge, obtains 2.38g, sample presentation detection, conversion ratio 88.94% by 1 method of embodiment extraction crude product.Crude product 0.8-1.5
After times volume ethyl acetate dissolved clarification, 5-10 times of twice of dilution crystallization of volume hexamethylene purification is added, obtains fine work 1.99g, liquid phase contains
Amount 99.7%
Embodiment 3
Seed liquor shaking flask is prepared, glucose 4.5%, corn pulp 2.0%, adjusting pH value is 4- 4.5.Use 500 milliliter three
Angle bottle, 100 milliliters of liquid amount, 115 DEG C sterilize for 15 minutes, after being cooled to room temperature, an oese saccharomyces uvarum are accessed, in 30 DEG C
200 rpms shaking table culture 16 hours；Prepare fermentation liquid shaking flask, glucose 5.5%, corn pulp 2.0%, yeast extract
0.9%, potassium dihydrogen phosphate 0.68%, sodium hydroxide 0.13%, pH6.2-6.4, with 500 milliliters of triangular flasks, 100 milli of liquid amount
It rises, 115 DEG C sterilize for 15 minutes.It is cooled to room temperature.
Every fermentation liquid shaking flask moves into 20 milliliters of strain liquid, crushes in 30 DEG C of 200 rpms of shaking table cultures, 14 hours investments
Isosorbide-5-Nitrae-androstenedione 0.25 of 100 meshes is smashed it through after 0.25 gram, 12 hours of the Isosorbide-5-Nitrae-androstenedione sieved with 100 mesh sieve afterwards again
Gram, while nutritive dextrose 1% is added, yeast extract 0.3%, investment smashes it through Isosorbide-5-Nitrae-hero of 100 meshes again after 12 hours
0.25 gram of alkene diketone, nutrition is equally added, it is primary to add within every 12 hours nutrition later, until the discharging that heats up after 96 hours.5 are shaken
Bottle extracts crude product by 1 method of embodiment, obtains 3.62 crude products, conversion ratio 90.99%.0.8-1.5 times of volume ethyl acetate of crude product is molten
After clear, 5-10 times of twice of dilution crystallization of volume hexamethylene purification is added, obtains fine work 2.96g, liquid content 99.6%
Embodiment 4
Seed liquor shaking flask is prepared, glucose 4.5%, corn pulp 2.0%, adjusting pH value is 4- 4.5.Use 500 milliliter three
Angle bottle, 100 milliliters of liquid amount, 115 DEG C sterilize for 15 minutes, after being cooled to room temperature, an oese saccharomyces cerevisiae are accessed, in 30 DEG C
200 rpms shaking table culture 16 hours；Prepare fermentation liquid shaking flask, glucose 5.5%, corn pulp 2.0%, yeast extract
0.9%, potassium dihydrogen phosphate 0.68%, sodium hydroxide 0.13%, pH6.2-6.4, with 500 milliliters of triangular flasks, 100 milli of liquid amount
It rises, 115 DEG C sterilize for 15 minutes.It is cooled to room temperature.
Every fermentation liquid shaking flask moves into 20 milliliters of strain liquid, in 30 DEG C of 200 rpms of shaking table cultures 14 hours investments, 2 times of second
0.25 gram of Isosorbide-5-Nitrae-androstenedione of 2 times of ethyl alcohol dissolution is put into after 0.25 gram of Isosorbide-5-Nitrae-androstenedione, 12 hours of alcohol dissolution again, together
When add nutritive dextrose 1%, yeast extract 0.3% puts into Isosorbide-5-Nitrae-androstenedione of 2 times of ethyl alcohol dissolution again after 12 hours
0.25 gram, while adding nutritive dextrose 1%, yeast extract 0.3%, it is primary to add within every 12 hours nutrition later, until 127 hours
Heating discharging afterwards extracts 5 bottles of shaking flask filter cakes by 1 method of embodiment, obtains crude product 3.48g, conversion ratio 92.43%.Crude product 0.8-1.5
After times volume ethyl acetate dissolved clarification, 5-10 times of twice of dilution crystallization of volume hexamethylene purification is added, obtains fine work 2.75g, liquid phase contains
Amount 99.3%.
Embodiment 5
Seed liquor shaking flask is prepared, glucose 4.5%, corn pulp 2.0%, adjusting pH value is 4- 4.5.Use 500 milliliter three
Angle bottle, 100 milliliters of liquid amount, 115 DEG C sterilize for 15 minutes, after being cooled to room temperature, an oese saccharomyces cerevisiae are accessed, in 30 DEG C
200 rpms shaking table culture 16 hours；Prepare fermentation liquid shaking flask, glucose 5.5%, corn pulp 2.0%, yeast extract
0.9%, potassium dihydrogen phosphate 0.68%, sodium hydroxide 0.13%, pH6.2-6.4, with 500 milliliters of triangular flasks, 100 milli of liquid amount
It rises, 115 DEG C sterilize for 15 minutes.It is cooled to room temperature.
Every fermentation liquid shaking flask moves into 20 milliliters of strain liquid, in 30 DEG C 200 rpms of shaking table cultures 14 hours, by fermentation liquid
It is centrifuged 5 minutes, discards supernatant under 3000G centrifugal force, be centrifuged after washing is resuspended with the PBS buffer solution of 10 milliliters of pH6.4, abandoned
Supernatant is removed, after being repeated twice, thallus is resuspended with the PBS buffer solution of the pH6.4 isometric with fermentation liquid, investment smashes it through 100
It feeds intake again 0.25 gram after 0.25 gram, 12 hours of Isosorbide-5-Nitrae-androstenedione of mesh, while filling into glucose 1%, every 12 is small later
When fill into glucose 1%, until conversion 72 hours after, heating terminate fermentation.5 bottles of shaking flasks merge, with the extraction side in demonstration example 1
Method is extracted, and crude product 2.43g, conversion ratio 86.98% are obtained.After 0.8-1.5 times of volume ethyl acetate dissolved clarification of crude product, 5-10 times of body is added
Product hexamethylene dilution crystallization refines 3 times, obtains fine work 1.65g, liquid content 99.8%.
Industrial applicibility
The method that the present invention provides a kind of to prepare boldenone by way of microorganism conversion.With Isosorbide-5-Nitrae-androstenedione
(ADD) it is raw material, 17 ketone groups using microbial selective is reduced to β hydroxyl to prepare boldenone (BD).This method benefit
The method for preparing boldenone with microorganism conversion avoids chemical reducing agent high using risk in production storage and transportation, converts item
Part is mildly environmentally friendly, and process safety is high, at low cost, is suitble to industrialized production.
Claims (9)
1. a kind of method for preparing boldenone, which is characterized in that convert Baodan for Isosorbide-5-Nitrae-androstenedione (ADD) using saccharomycete
Ketone, the saccharomycete are saccharomyces cerevisiaesSaccharomyces cerevisiaeCGMCC 2.346 or saccharomyces uvarumSaccharomyces uvarum One of CGMCC 2.4636, conversion process such as following formula:
。
2. preparing the method for boldenone according to claim 1, which is characterized in that the feeding mode of fermentation is that micronization is thrown
Material, micronization Isosorbide-5-Nitrae-androstenedione sieve with 100 mesh sieve net.
3. preparing the method for boldenone according to claim 2, which is characterized in that transform mode includes direct plungeing into fermentation liquid
Conversion.
4. preparing the method for boldenone according to claim 2, which is characterized in that transform mode includes that building resting cell turns
The conversion of change system.
5. preparing the method for boldenone according to claim 1, which is characterized in that the feeding mode of fermentation is that solvent dissolution is thrown
Material, the solvent is ethyl alcohol or isopropanol.
6. preparing the method for boldenone according to claim 5, which is characterized in that transform mode includes direct plungeing into fermentation liquid
Conversion.
7. preparing the method for boldenone according to claim 5, which is characterized in that transform mode includes that building resting cell turns
The conversion of change system.
8. preparing the method for boldenone according to claim 1, which is characterized in that the Isosorbide-5-Nitrae-androstenedione is to throw by several times
Material, total feed concentrations of step feeding are 0.5-0.75%.
9. preparing the method for boldenone according to claim 1, which is characterized in that conversion process needs to add nutrients by several times
Matter, including carbon source and nitrogen source；The carbon source material includes: glucose；The nitrogen source includes: yeast extract.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114292892A (en) * | 2022-01-05 | 2022-04-08 | 浙江仙琚制药股份有限公司 | Method for producing androstadienedione by virtue of Arthrobacter simplex fermentation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108085359A (en) * | 2016-11-22 | 2018-05-29 | 保定九孚生化有限公司 | A kind of production method of 11 alpha-hydroxy-4-ene -3,17- androstanediones |
-
2019
- 2019-01-16 CN CN201910037611.6A patent/CN109852658B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108085359A (en) * | 2016-11-22 | 2018-05-29 | 保定九孚生化有限公司 | A kind of production method of 11 alpha-hydroxy-4-ene -3,17- androstanediones |
Non-Patent Citations (2)
| Title |
|---|
| MOHAMMAD ALI FARAMARZI等: "Nostoc muscorum: a regioselective biocatalyst for 17-carbonyl reduction of androst-4-en-3,17-dione and androst-1,4-dien-3,17-dione", 《ANNALS OF MICROBIOLOGY》 * |
| YOSSI SINGER等: "Microbial transformations in a cyclodextrin medium. Part 2. Reduction of androstenedione to testosterone by Saccharomyces cerevisiae", 《APPL MICROBIOL BIOTECHNOL》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114292892A (en) * | 2022-01-05 | 2022-04-08 | 浙江仙琚制药股份有限公司 | Method for producing androstadienedione by virtue of Arthrobacter simplex fermentation |
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| CN109852658B (en) | 2021-08-27 |
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