CN101525651A - Method for preparing androstenedione by biodegradation of phytosterol in two-liquid-phase system - Google Patents
Method for preparing androstenedione by biodegradation of phytosterol in two-liquid-phase system Download PDFInfo
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Abstract
The invention relates to a bio-pharmaceutical preparation, in particular to a method for preparing androstenedione by biodegradation of phytosterol in a two-liquid-phase system. The microbial selective side-chain degradation of phytosterol is carried out to obtain an important intermediate androstenedione synthesized by steroid drugs, and the androstenedione is of great significance to the pharmaceutical industry in China. In the two-liquid-phase system, the process for preparing the AD by biodegradation of phytosterol and the influences of substrate input amount, the surface active agent and the added inhibitor to degradation are studied. The result shows that the sunflower oil is the optimum organic solvent for preparing the AD by degradation of phytosterol by mycobacteria, and the transformation is influenced by the substrate input amount and the added hydroxylase inhibitor. The optimum transformation condition of the sunflower oil/aqueous system is determined through the orthogonal test: the concentration of the sunflower oil is 20 percent, the substrate input amount is 0.4 percent, and the additive agent is 0.005 percent of mercury bichloride, and the AD conversion rate is up to 83.4 percent under the process condition.
Description
Technical field
The present invention relates to a kind of biological medicine company preparation, particularly relate to microbial selective degrading plant sterol side chain and obtain steroid drugs synthetic important intermediate " Androstenedione ".The invention still further relates to this microbial selective degrading plant sterol side chain and obtain the preparation method of steroid drugs synthetic important intermediate " Androstenedione ".
Technical background
Androstane-4-alkene-3, the 17-diketone (androst-4-ene-3,17-dione) be called for short " Androstenedione " or " AD ", be the irreplaceable intermediate of steroid hormone class medicine, body is played very important regulating effect.China's production " AD " at present mainly is to extract diosgenin to obtain through chemosynthesis from wild Chinese medicinal materials plants such as " Dioscorea nipponica Mak. Ningpo Yam Rhizomes ", and its technology is very complicated, and consuming cost is very high, but also contaminate environment.Extract " AD " by microbial fermentation; its suitability for industrialized production technology has tempting prospect; not only can fundamentally change weak points such as traditional chemical synthesizing steroid medicine step is many, yield is low, price is expensive; also can make full use of and bring into play resources advantage; break away from the starting material source because of of the restriction of natural causes such as season, region, to protecting national resource with ecotope, safeguarding the big effect of human health lifting to producing.
The agent structure of plant sterol and steroid hormone medicine all is a steroid nucleus, different just side chains and the difference on two key.These constructional features of plant sterol have determined it to can be used as the semisynthetic raw material of steroid hormone medicine.But, utilize chemical means degrading plant sterol side chain difficulty bigger, for a long time plant sterol is used as waste treatment, cause artificial waste resource.The development of microbial selective degradation of steroid side chain technology makes full use of these sterols cheap and easy to get becomes possibility.Microbial method is produced " AD " and mainly is subjected to the low restriction of sterol solubleness in water.Traditional single water fermentation methods of adding a small amount of organic solvent, tensio-active agent that adopt improve the concentration of sterol in fermented liquid more, but effect is unsatisfactory.Superpolymer/superpolymer aqueous two-phase system is applied to the microbial transformation that the sterol side chain excises because of good biocompatibility, but the solubilising of superpolymer is limited and price is more expensive, has limited its application in industry.Organic solvent/water two liquid phase systems solubilisings increase, research is more in the microbial transformation of sterol side chain excision, the main drawback of this system is the toxicity of organic solvent pair cell and the volatility of organic solvent, increased the complicacy of environment, safety etc.Biological food institute of HeFei University of Technology is by various mutagenesis obtain the superior strain that a strain can be converted into plant sterol " AD " to mycobacterium Mycobacterium sp.BD696#.This experiment proposes to substitute organic solvents such as ethanol commonly used, acetone, ethyl acetate with vegetables oil, not only can increase substrate solubleness, and reduce cytotoxicity.Main research mutant strain is in Trisun Oil R 80/water biliquid phase system, and the plant sterol microbial transformation prepares substrate charging capacity, tensio-active agent and the influence of interpolation inhibitor to transforming in the novel process of Androstenedione and the system.
Effective constituent and chemical property
Androstenedione
1) English popular name: Androst-4-ene-3,17-dione (4-AD)
2) chemical name: androstane-4-alkene-3,17-diketone
3) structural formula:
4) molecular formula: C
19H
26O
2
5) molecular weight: 286
6) physico-chemical property: outward appearance is accurate white crystalline powder, free from extraneous odour, and nontoxicity is dissolved in ethanol, acetic acid, ether, and is water insoluble, is the extremely wide biochemical industry intermediate of a kind of purposes.
The advantage of this invention:
1, extracts " AD " by microbial fermentation; its suitability for industrialized production technology has tempting prospect; not only can fundamentally change weak points such as traditional chemical synthesizing steroid medicine step is many, yield is low, price is expensive; also can make full use of and bring into play resources advantage; break away from the starting material source because of of the restriction of natural causes such as season, region, to protecting national resource with ecotope, safeguarding the big effect of human health lifting to producing.
2, the present invention's proposition is a Trisun Oil R 80 with the organic solvent, its transformation efficiency is the highest, and almost there is not similar by product androstane-1,4-diene-3, the generation of 17-diketone (ADD), Trisun Oil R 80 has emulsifying capacity preferably, and the generation of emulsion can further promote contacting of sterol and cell, thereby promote the conversion of sterol, and reduced the toxicity of organic solvent pair cell greatly.
Summary of the invention
The purpose of this invention is to provide a kind of effectively, cost is low, and the microbial selective degrading plant sterol side chain of safety and environmental protection obtains steroid drugs synthetic important intermediate " Androstenedione ".Another object of the present invention provides the preparation method of biodegradation of phytosterol " Androstenedione " in a kind of biliquid phase system.
The present invention realizes by following technical measures:
1 preparation materials and methods
1.1 material
1.1.1 bacterial classification
Bacterial classification source: Mycobacterium sp-UV-8 (the biological and Foodstuffs Academy preservation of HeFei University of Technology).
1.1.2 instrument and analysis condition
TLC: thin plate is aluminium foil silica gel 60F254 (a merck company), and moving phase is sherwood oil: ethyl acetate=6: 4, saturated 30min, exhibition layer 0.5h.Reagent: androstane-4-alkene-3,17-diketone (AD) are chromatographically pure (sigma company); All the other reagent are analytical pure.GC-MS: Tianjin, island GC-14 gas chromatography system.Chromatographic condition: chromatographic column is that (4mm * 2mm), column temperature adopts temperature programming to the stainless steel column of PEG-20M, and initial column temperature is kept 3min for 210 ℃, and the speed with 30 ℃/min rises to 280 ℃ again, keeps 10min.Content calculates with the external standard peak area method.
1.2 implementation method
1.2.1 substratum
Slant medium (M1, %): glucose 1; Yeast extract paste 0.5; Polyprotein peptone 0.5; Agar powder 2, KH
2PO
40.1, NH
4NO
30.1, pH7.0～7.2.
Seed culture medium (M2, %): molasses 6.0, glucose 0.8, KH
2PO
40.06, NH
4NO
30.5 back pH 7.0～7.2 disappears.
The conversion substratum (M3, %): molasses 6.0, sterol 1, glycerine 16, KH
2PO
40.06, NH
4NO
30.5, pH8.2～8.4.
1.2.2 culture condition
Inclined-plane: 30 ℃～32 ℃ of temperature, growth cycle 5d; Separating plate: 5～7d.
Shake-flask seed: 30 ℃～32 ℃ of temperature, rotating speed 130r/min, growth cycle 72h.
Transform: inoculum size 10%, 30 ℃～32 ℃ of temperature, rotating speed 250～280r/min, growth cycle 120～140h.
1.2.3 technology of preparing
(1) system that different concentration ethanol/water, glycerin/water, Trisun Oil R 80/water are formed carries out organic solvent/water two-phase system transformation experiment, selects best organic solvent.
(2) various additives are to the influence of system
On the basis of above implementation method, in system, add different concns tensio-active agent (tween-80), inhibitor (oxine, 2,2 '-dipyridyl, mercury chloride etc.) respectively, study of the influence of various additives to transformation efficiency.
1.2.4 the method for calculation of plant sterol transformation efficiency
After fermentation is finished, extract 4AD with ethyl acetate with 1: 1 ratio, dilute 100 times then, quantitatively measure the content of Androstenedione again with gas-chromatography, the following formula of utilization calculates the transformation efficiency of plant sterol then: AD content * 100%/(sterol content in the oil that feeds intake * 0.66 * 0.95) in transformation efficiency=purification back organic phase.
2 results and discussion
2.1 organic solvent is to the influence of transformation efficiency
In the sterol biotransformation, solvent separates solubleness, cytoactive and the product of substrate and product all very big influence.Therefore, when making up two liquid-phase systems, most critical be choice of Solvent.
Find out by table 1, when organic solvent is Trisun Oil R 80, transformation efficiency is the highest, and almost there is not similar by product androstane-1,4-diene-3, the generation of 17-diketone (ADD), Trisun Oil R 80 has emulsifying capacity preferably, the generation of emulsion can further promote contacting of sterol and cell, thereby promotes the conversion of sterol, and has reduced the toxicity of organic solvent pair cell greatly; Glycerine is during as organic solvent, has under the lower concentration outside a small amount of " AD " generate, and all has " ADD " to generate under the high density, is that later separation increases difficulty; When organic solvent was ethanol, transformation efficiency was also lower, and the highest reaches 36.8%, and often with the generation of " ADD ".
The result shows: in Trisun Oil R 80/water system, Trisun Oil R 80 concentration is 20%, 30% and all can reach higher transformation efficiency at 40% o'clock, and oil phase concentration is 10% o'clock, and emulsification does not take place substantially, and reason is that oil phase concentration is less, and sterol can not well dissolve.The conversion of sterol is unsatisfactory in the glycerin/water system, except having under lower concentration " AD " generate, all do not have " AD " generation under other concentration, and the generation of " ADD " is generally arranged in each concentration.Illustrate that the glycerin/water system helps sterol more and is converted into " ADD ".In the ethanol/water system, the increase of alcohol concn can promote the conversion of sterol, and the transformation efficiency of sterol is the highest when alcohol concn is 4%, sharply descends and continue to increase to 5% o'clock sterol transformation efficiency.Reason is an ethanol consumption very little the time, and the substrate sterol can not finely dissolve; When the ethanol consumption is too many,, influence the growth of thalline, suppress the side chain degrading enzymatic activity, be unfavorable for transforming then to the thalline toxigenicity.Therefore determine that Trisun Oil R 80 is best organic solvent.
The influence of table 1 organic solvent/water two-phase system to transforming
Tab.1effect of organic/water biphasic systems on bioconversion
2.2 the substrate charging capacity is to the influence of transformation efficiency
Sterol is a kind of hydrophobicity crystallization, solubleness in water extremely low (being usually less than 0.1mmol/l), and aborning in order to improve transformation efficiency, transform under the concentration conditions that often is higher than substrate solubleness and carry out far away, this excessive substrate certainly will produce thalline and poison, be unfavorable for the carrying out that sterol transforms, and cause raw-material waste.Trisun Oil R 80/water system preferably resolves this problem.
By Fig. 1 substrate charging capacity as can be known to the influence that transforms, increase with the substrate charging capacity, transformation efficiency is in rising trend gradually, transformation efficiency rises to about 79% when charging capacity increases to 0.5%, transformation efficiency begins to descend then, and particularly concentration of substrate was near 0.6% o'clock, and transformation efficiency sharply descends, reason is the dissolved oxygen that excessive substrate has influence on fermention medium, thereby suppresses thalli growth.
2.3 additive is to the influence of transformation efficiency
2.3.1 the influence of tensio-active agent to transforming
In theory, in the biotransformation, tensio-active agent increases the solubleness of substrate on the one hand, changes the permeability of cytolemma simultaneously, should be able to produce appreciable results to the transmission of quickening substrate with action of microorganisms.Tween-80 has another name called Tween 80, is the comparatively general tensio-active agent of a kind of application, is oil-in-water emulsifiers, can be used as the solubilizing agent of insoluble medicine in some water.
By Fig. 2 tween-80 to the influence that transforms as can be known, the concentration of tween-80 is higher at 2% o'clock transformation efficiency, reaches 60.5%, along with the increase transformation efficiency of concentration descends gradually.Transformation efficiency all is lower than not added-time transformation efficiency (74.1%) under each concentration, and reason is because the adding pair cell of tween-80 has produced toxicity, thereby has suppressed the carrying out that transforms.Therefore, though tensio-active agent has solubilising, effect and not obvious in Trisun Oil R 80/water biliquid phase system.
2.3.2 the influence of hydroxylase inhibitors to transforming
Sterol transforms in the process of " AD ", simultaneously can be with the further degraded of " AD ", and degradation production is water and CO
2, this reaction is to carry out under the effect of 9 α-hydroxylase, suppress the degraded of " AD ", must suppress the activity of 9 α-hydroxylase.9 α-hydroxylase is a kind of mono-oxygenase that contains ferrous ion, removes or replace ferrous ion to cause its active forfeiture.Fe
2+Sequestrant oxine (8-HQ) and 2, and 2 '-dipyridyl (2,2-Dipyridyl) be 9 α-hydroxylase inhibitors of using always.In addition, Hg
2+It also is 9 α-hydroxylase inhibitors of using always.
9 α-hydroxylase generally adds at the thalline logarithmic phase, and this moment, thalli growth was basicly stable, and what carry out in system mainly is conversion to sterol, and adding 9 α-hydroxylase inhibitors may produce favourable influence to transforming.When cultivating beginning, experimental selection adds and logarithmic phase (72h after the fermentation culture) adding, as shown in Table 2, fermentation culture begins just to add 9 α-hydroxylase inhibitors, add 8-HQ and 2, all do not have the generation of " AD " in the fermention medium of 2 '-Dipyridyl, have only a spot of AD to produce in the substratum of adding mercury chloride yet.Effect has taken place at the very start in the toxicity that may be each inhibitor, causes the cell poor growth, has influenced the conversion of sterol, and 8-HQ and 2, the toxicity of 2 '-Dipyridyl is stronger than the toxicity of mercury chloride.
72h after fermentation culture (logarithmic phase) adds 9 α-hydroxylase inhibitors, and when each inhibitor concentration was low, the adding of inhibitor helped sterol and improves transformation efficiency, 8-HQ, 2,2-Dipyridyl, HgCL
2Concentration was at 0.005% o'clock, transformation efficiency reaches 80.5%, 76.4%, 73.6% respectively, in the time of more unconstrained dose, transformation efficiency all increases, and when concentration increases, transforms decline to some extent, may be because each inhibitor concentration is big more, the toxicity of its pair cell is just big more, though after cultivating 72h, add, but played adverse influence.Illustrate under 9 α-hydroxylase lower concentration and be beneficial to conversion.
2.4 orthogonal method
On the basis of above single factor scheme, choose oil phase concentration (%), charging capacity (%), inhibitor (%) as investigating index, adopts the design of three factors, three horizontal quadrature methods with the AD yield as the investigation factor.Level of factor, experimental design and the results are shown in Table 3
2.4.1 scheme and interpretation of result
As shown in Table 3, each factor affecting from the master to inferior order is: A (oil phase concentration), B (charging capacity), C (inhibitor).All greater than the extreme difference of sky row, hence one can see that, and each factor all has certain influence to the result to the extreme difference R of each factor.Preferred embodiment is A
2B
1C
3, promptly oil phase concentration 20%, charging capacity 0.4%, and inhibitor is 0.005% mercury chloride, this moment, peak rate of conversion was 83.4%, exceeded 3.4 percentage points than before peak rate of conversion 80%.
Find out that from the table 4 as a result of variance analysis organic solvent concentration, substrate charging capacity are all influential to transformation efficiency, but not remarkable.This is because in Trisun Oil R 80/water biliquid phase system, substrate and product mainly exist in organic phase, and there is water in inhibitor, therefore suppress the further degradation effect of product and not obvious, from foreign data, adding inhibitor in general single water system or the superpolymer/superpolymer aqueous two-phase system can play a role.
Table 3 orthogonal scheme and interpretation of result
Tab.3 Design and result of orthogonal test L
9(3
4)
The variance analysis of table 4 quadrature
Tab.4 The analysis of variance of results of orthogonal test
| Soruces of variation | S | f | V | The F value | |
| A B C e | 51.709 46.882 23.631 15.416 | 2 2 2 2 | 25.855 23.441 11.816 7.708 | 3.35 3.04 1.53 | |
| Summation T | 137.638 | 8 |
3 conclusions
(1) prepare in the AD technology at mycobacterium degrading plant sterol, Trisun Oil R 80 not only can increase sterol solubleness as organic solvent, and can reduce the toxicity of organic solvent pair cell.
(2) Trisun Oil R 80/water biliquid phase system is the best system of changing effect in this research, and transformation efficiency can reach 80%, exceeds about 20% than other system's transformation efficiency.In this system, problems such as the solvent toxicity that the sterol conversion upward exists, substrate dissolving, product inhibition have been solved substantially.
(3) though tensio-active agent has solubilising, effect and not obvious in Trisun Oil R 80/water biliquid phase system.
(4) hydroxylase inhibitors is favourable to transforming when lower concentration, during high density, because the toxicity of pair cell is unfavorable for transforming.
(5) by orthogonal experimental design, obtained the sterol transformation efficiency and be 83.4% reactive system: Trisun Oil R 80/water biliquid phase system, wherein Trisun Oil R 80 concentration 20%, substrate charging capacity 0.4%, additive is that concentration is 0.005% mercury chloride.
Claims (7)
1, a kind of biological medicine company preparation, specifically " method of biodegradation of phytosterol preparation " Androstenedione " in a kind of biliquid phase system ".
2, obtain the preparation method of steroid drugs synthetic important intermediate " Androstenedione " according to claim 1 described microbial selective degrading plant sterol side chain.
3, obtain the substratum of steroid drugs synthetic important intermediate " Androstenedione " according to claim 1 described microbial selective degrading plant sterol side chain.Slant medium (M1, %): glucose 1; Yeast extract paste 0.5; Polyprotein peptone 0.5; Agar powder 2, KH
2PO
40.1, NH
4NO
30.1, pH7.0～7.2; Seed culture medium (M2, %): molasses 6.0, glucose 0.8, KH
2PO
40.06, NH
4NO
30.5 back pH 7.0～7.2 disappears; The conversion substratum (M3, %): molasses 6.0, sterol 1, glycerine 16, KH
2PO
40.06, NH
4NO
30.5, pH8.2～8.4.
4, obtain the culture condition of steroid drugs synthetic important intermediate " Androstenedione " according to claim 1 described microbial selective degrading plant sterol side chain.Inclined-plane: 30 ℃～32 ℃ of temperature, growth cycle 5d; Separating plate: 5～7d; Shake-flask seed: 30 ℃～32 ℃ of temperature, rotating speed 130r/min, growth cycle 72h; Transform: inoculum size 10%, 30 ℃～32 ℃ of temperature, rotating speed 250～280r/min, growth cycle 120～140h.
5, obtain the technology of preparing of steroid drugs synthetic important intermediate " Androstenedione " according to claim 1 described microbial selective degrading plant sterol side chain.(1) system that different concentration ethanol/water, glycerin/water, Trisun Oil R 80/water are formed carries out organic solvent/water two-phase system transformation experiment, selects best organic solvent; (2) obtain of the influence experiment of the various additives of steroid drugs synthetic important intermediate " Androstenedione " according to claim 1 described microbial selective degrading plant sterol side chain to system, on above experimental basis, in system, add different concns tensio-active agent (tween-80), inhibitor (oxine respectively, 2,2 '-dipyridyl, mercury chloride etc.), study of the influence of various additives to transformation efficiency.
6, obtain the detection method of the plant sterol transformation efficiency of steroid drugs synthetic important intermediate " Androstenedione " according to claim 1 described microbial selective degrading plant sterol side chain.Tianjin, island GC-14 gas chromatography system. chromatographic condition: chromatographic column is the stainless steel column (4 * 2mm) of PEG-20M, column temperature adopts temperature programming, initial column temperature is kept 3min for 210 ℃, and the speed with 30 ℃/min rises to 280 ℃ again, keeps 10min. content and calculates with the external standard peak area method.
7, obtain the method for calculation of the plant sterol transformation efficiency of steroid drugs synthetic important intermediate " Androstenedione " according to claim 1 described microbial selective degrading plant sterol side chain.After fermentation is finished, extract 4AD with ethyl acetate with 1: 1 ratio, dilute 100 times then, quantitatively measure the content of Androstenedione again with gas-chromatography, the following formula of utilization calculates the transformation efficiency of plant sterol then: AD content * 100%/(sterol content in the oil that feeds intake * 0.66 * 0.95) in transformation efficiency=purification back organic phase.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102061320A (en) * | 2010-12-02 | 2011-05-18 | 浙江仙琚制药股份有限公司 | Preparation method of 11 alpha,17 alpha-dyhydroxyl-androst-4-ene-3,20-dione |
| CN102477404A (en) * | 2010-11-24 | 2012-05-30 | 北大方正集团有限公司 | Method for producing ADD from phytosterols by microbial transformation and culture medium thereof |
| CN102586139A (en) * | 2012-01-20 | 2012-07-18 | 广东本科生物工程股份有限公司 | High-yield AD/ADD strain and method for high-efficient production of AD/ADD |
| CN103613627A (en) * | 2013-11-20 | 2014-03-05 | 上海市农药研究所 | Method for recycling edible oil in androstenedione production process through biotransformation |
| CN104560765A (en) * | 2013-10-20 | 2015-04-29 | 山东方明药业集团股份有限公司 | Method for obtaining mycobacterium strains with resistance |
| CN104651261A (en) * | 2014-12-08 | 2015-05-27 | 云南中烟工业有限责任公司 | Microbial strain and applications thereof in degradation of tobacco sterol |
| CN105567599A (en) * | 2016-01-26 | 2016-05-11 | 山东赛托生物科技股份有限公司 | Efficient breeding method of 4-androstenedione strains |
| CN105861612A (en) * | 2016-04-27 | 2016-08-17 | 天津科技大学 | Preparation method of androstenedione |
| CN105907828A (en) * | 2016-04-27 | 2016-08-31 | 天津科技大学 | Substrate treatment method for phytosterol side chain degradation reaction |
| CN109355345A (en) * | 2018-11-22 | 2019-02-19 | 安徽建筑大学 | It is a kind of to synthesize androstane-4-alkene-3, the method for 7- diketone using mycobacteria degrading plant sterol |
| CN110656147A (en) * | 2019-10-16 | 2020-01-07 | 湖南新合新生物医药有限公司 | Biological dehydrogenation method of androstenedione C1,2 site |
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| CN102477404A (en) * | 2010-11-24 | 2012-05-30 | 北大方正集团有限公司 | Method for producing ADD from phytosterols by microbial transformation and culture medium thereof |
| CN102477404B (en) * | 2010-11-24 | 2014-04-02 | 北大方正集团有限公司 | Method for producing ADD from phytosterols by microbial transformation and culture medium thereof |
| CN102061320B (en) * | 2010-12-02 | 2013-04-03 | 浙江仙琚制药股份有限公司 | Preparation method of 11 alpha,17 alpha-dyhydroxyl-androst-4-ene-3,20-dione |
| CN102061320A (en) * | 2010-12-02 | 2011-05-18 | 浙江仙琚制药股份有限公司 | Preparation method of 11 alpha,17 alpha-dyhydroxyl-androst-4-ene-3,20-dione |
| CN102586139A (en) * | 2012-01-20 | 2012-07-18 | 广东本科生物工程股份有限公司 | High-yield AD/ADD strain and method for high-efficient production of AD/ADD |
| CN102586139B (en) * | 2012-01-20 | 2013-03-20 | 广东本科生物工程股份有限公司 | High-yield AD/ADD strain and method for high-efficient production of AD/ADD |
| CN104560765A (en) * | 2013-10-20 | 2015-04-29 | 山东方明药业集团股份有限公司 | Method for obtaining mycobacterium strains with resistance |
| CN103613627B (en) * | 2013-11-20 | 2017-12-01 | 上海市农药研究所 | The recycling method of food-grade oil during bioconversion production androstenedione |
| CN103613627A (en) * | 2013-11-20 | 2014-03-05 | 上海市农药研究所 | Method for recycling edible oil in androstenedione production process through biotransformation |
| CN104651261A (en) * | 2014-12-08 | 2015-05-27 | 云南中烟工业有限责任公司 | Microbial strain and applications thereof in degradation of tobacco sterol |
| CN104651261B (en) * | 2014-12-08 | 2017-09-22 | 云南中烟工业有限责任公司 | A kind of microbial strains and its application in degrading tobacco sterol |
| CN105567599A (en) * | 2016-01-26 | 2016-05-11 | 山东赛托生物科技股份有限公司 | Efficient breeding method of 4-androstenedione strains |
| CN105861612A (en) * | 2016-04-27 | 2016-08-17 | 天津科技大学 | Preparation method of androstenedione |
| CN105907828A (en) * | 2016-04-27 | 2016-08-31 | 天津科技大学 | Substrate treatment method for phytosterol side chain degradation reaction |
| CN105861612B (en) * | 2016-04-27 | 2019-04-05 | 天津科技大学 | A kind of preparation method of androstenedione |
| CN105907828B (en) * | 2016-04-27 | 2019-07-05 | 天津科技大学 | A kind of substrate processing method for the reaction of phytosterol Side chain cleavage |
| CN109355345A (en) * | 2018-11-22 | 2019-02-19 | 安徽建筑大学 | It is a kind of to synthesize androstane-4-alkene-3, the method for 7- diketone using mycobacteria degrading plant sterol |
| CN110656147A (en) * | 2019-10-16 | 2020-01-07 | 湖南新合新生物医药有限公司 | Biological dehydrogenation method of androstenedione C1,2 site |
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