CN101191141B - Fermentation culture medium for biologically synthesizing 7alpha, 15alpha-dihydroxyandrostenolone - Google Patents
Fermentation culture medium for biologically synthesizing 7alpha, 15alpha-dihydroxyandrostenolone Download PDFInfo
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- CN101191141B CN101191141B CN2006101185104A CN200610118510A CN101191141B CN 101191141 B CN101191141 B CN 101191141B CN 2006101185104 A CN2006101185104 A CN 2006101185104A CN 200610118510 A CN200610118510 A CN 200610118510A CN 101191141 B CN101191141 B CN 101191141B
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Abstract
The invention discloses a fermentation medium for biosynthesis of 7 alpha-dihydroxy androstene alcohol ketone and 15 alpha-dihydroxy and rostene alcohol ketone by taking flax colletotrichum (Colletotrichum lini AS 3.4) as strain, consisting of organic nitrogen sources, carbon sources, inorganic salts and metal ions. The invention is characterized in that: the organic nitrogen sources are peptones, yeast extracts, beef extracts and yeast extract pastes. When a product I is synthesized by adoption of the fermentation medium of the invention, substrate feed concentration can reach 8 grams per liter (substrate weight/fermentation broth volume) and conversion ratio can reach 85 percent, thereby the invention can well solve the defects of low substrate feed concentration and low conversion ratio of the prior art, and then industrial production cost of the product I is reduced.
Description
Technical field
The present invention relates to the mikrobe steroidal and transform substratum, be specifically related to a kind of biosynthesizing 7 α, fermentation culture medium for microbe of 15 alpha-dihydroxy-dihydrotestosterones of being used for.
Background technology
Steroidal class midbody often can be used for producing various clinically medicines.7 α, 15 alpha-dihydroxy-dihydrotestosterones (its structure is as shown in the formula I)
Be a kind of important steroidal midbody, can be used for multiple medicines such as synthetic lactone aldosterone receptor antagonist, diuretic(s) and gynecologic medicine.For example, I is the important intermediate of synthetic oral contraceptive Yasmin activeconstituents drospirenone.
Microbial transformation has critical role in steroid drugs synthetic, the important method of production steroid hormone and analogue thereof are gone up in several kinds of important steroidal microbial transformations reactions as hydroxylation, dehydrogenation and side chain degraded having become industry.The preparation of compound I is difficult to the method for chemistry synthetic, and is mainly synthetic by microbial transformation at present.
I can be synthetic through microbial transformation by dehydroepiandros-sterone (its structure is as shown in the formula II).It is the method that bacterial classification is converted into II I that Kieslich (German Patent No.DE 2746298 1979-04-19) has described with capsicum thorn dish spore (Colletotrichum phomoides ifo 5257); And (Angew.Chem.95 (5), 413-414,1983 such as Petzoldt; U.S.Patent No.44353271984-03-06) then having described with flax thorn dish spore (Colletotrichum lini cbs112.21) is the method that bacterial classification is converted into II I.But the concentration that feeds intake of two kinds of method substrates is very low, has only 1g/L, so transformation efficiency is very low, industrial cost is very high.Shang Ke etc. (Chinese medicine biotechnology applications magazine .2,30～34,2004) report flax thorn dish spores (Colletotrichum lini AS3.4486) can be converted into I with II; The very low 2g/L of having only of concentration but substrate feeds intake; Yield is low to have only 58.8%, and transformation efficiency is also lower, is about 70%.It is thus clear that the existing skill of changing a job can not be synthesized I efficiently, is badly in need of more efficiently conversion process, to improve the feed intake concentration and the transformation efficiency of substrate, to reduce production costs.
Summary of the invention
For solving the problems of the technologies described above, the purpose of this invention is to provide that a kind of (Colletotrichum lini AS3.4486 is preserved in Chinese common micro-organisms culture presevation administrative center with flax thorn dish spore; Numbering AS3.4486) be bacterial classification; Biosynthesizing 7 α, the fermention medium of 15 alpha-dihydroxy-dihydrotestosterones comprises organic nitrogen source, carbon source, inorganic salt and metals ion; It is characterized in that described organic nitrogen source is peptone, yeast extract, Carnis Bovis seu Bubali cream, yeast extract paste.
The content of described organic nitrogen source is 6g/L～30g/L, preferred 10g/L～15g/L, more preferably 12g/L.
Preferably, described organic nitrogen source is a yeast extract.
Described carbon source is the polyose carbon source.
Said polyose carbon source is dextrin, maltodextrin, starch, Zulkovsky starch, Semen Maydis powder etc., is preferably maltodextrin and Zulkovsky starch, more preferably maltodextrin; Its content is 20g/L～50g/L, is preferably 35g/L.
Fermention medium of the present invention can also contain natural fats and oils.
Said natural fats and oils can be soya-bean oil, rapeseed oil, corn embryo oil, sunflower seed oil and cottonseed wet goods vegetables oil; Animal oil and fat such as lard, sheep oil, butter and fish oil; And phosphatide such as Yelkin TTS, POPG, phosphatidic acid; Its content is 0.5g/L～10g/L, and more excellent is 2g/L～5g/L, and optimum is 3g/L.
Also need add metals ions such as inorganic salt such as sylvite, phosphoric acid salt, ammonium salt and nitrate salt and magnesium in the substratum.
Different organic nitrogen sources are very big to the influence that transforms; The effects groundnut meal, soybean cake powder, cottonseed meal, dregs of beans, peptone, yeast extract, Carnis Bovis seu Bubali cream and nine kinds of organic nitrogen sources commonly used of yeast extract paste; The concentration of adding is 10g/L; The concentration that feeds intake of substrate is identical, transforms to finish the concentration (seeing accompanying drawing 1) that back HPLC detects product in each organic nitrogen source fermented liquid.Can find out that from figure organic nitrogen source adopts peptone, yeast extract, Carnis Bovis seu Bubali cream, yeast extract paste to be the content height of product, wherein the highest with yeast extract again.
Different carbon sources also have tangible influence to transforming; The effects carbon sources such as dextrin, maltodextrin, starch, Zulkovsky starch, Semen Maydis powder, sucrose, SANMALT-S, glucose and fructose; The concentration of adding is 30g/L; The concentration that feeds intake of substrate is identical, transforms to finish the concentration (seeing accompanying drawing 2) that back HPLC detects product in each carbon source through fermentation liquid.From figure can find out polyose carbon sources such as dextrin, maltodextrin, starch, Zulkovsky starch and Semen Maydis powder as carbon source the time bacterial classification conversion capability stronger; And with disaccharides such as sucrose and SANMALT-S, or monose carbon sources such as glucose and fructose during as carbon source conversion capability then lower.
In substratum, add the growth that a spot of natural fats and oils can promote thalline; Better promote the conversion of substrate in fermented liquid simultaneously; Experiment has been chosen multiple natural fats and oils such as soya-bean oil, rapeseed oil, corn embryo oil, sunflower seed oil, lard, fish oil, Yelkin TTS and POPG and has been added substratum respectively; Add to measure and be 3g/L; As contrast, the concentration that feeds intake of substrate is identical with the substratum that do not add natural fats and oils, transforms to finish the concentration (seeing accompanying drawing 3) that back HPLC detects product in each substratum.After scheming to find out all kinds of natural fats and oils of adding, transformation efficiency all is significantly improved.
When adopting fermention medium synthetic product I of the present invention, the substrate concentration that feeds intake can reach 8g/L (substrate weight/fermentating liquid volume), and transformation efficiency can arrive 85%.And when adopting the fermention medium synthetic product I of prior art, the substrate concentration that feeds intake has only 2g/L, and transformation efficiency is about about 70%.Therefore, through the present invention through the fermention medium of constituent optimization carry out the conversion of I synthetic can solve well the prior art substrate feed intake concentration low with the not high shortcoming of transformation efficiency, thereby reduce the industrial production cost of I.
Description of drawings
Fig. 1 representes the influence of different organic nitrogen sources to conversion reaction;
Fig. 2 representes the influence of different carbon sources to conversion reaction;
Fig. 3 has represented to add the promoter action of the substratum of natural fats and oils to conversion reaction.
Embodiment
Below come further to illustrate the present invention through concrete embodiment, but these embodiment are not construed as limiting the invention.Wherein, Yeast extract is that Hubei Angel Yeast Co.,Ltd produces, and peptone is that the East Sea, Shanghai pharmaceutical factory produces, and Carnis Bovis seu Bubali cream is that West China, Chengdu biochemical product factory produces; Yeast extract paste, dextrin, starch, Zulkovsky starch, Yelkin TTS are that Shanghai chemical reagents corporation of Chinese Medicine group produces; Groundnut meal is that Beijing Kang Mingwei substratum technology Ltd produces, and maltodextrin is that sea salt six is produced with starch chemical industry ltd, and Semen Maydis powder is that Zhucheng abundant rich powder process ltd produces; Sunflower seed oil is that vegetables oil ltd produced in praised in Qingdao, and soya-bean oil is that grain and oil Industrial Co., Ltd produced in praised in Shanghai.
Substratum
Slant medium: PDA substratum (200g potato liquor 1000ml, glucose 20g, agar 20g), 121 ℃ of sterilization 20min.
Seed culture medium: groundnut meal 10g/L; Dextrin 30g/L; KCl 1g/L; (NH
4)
2HPO
41g/L; K
2HPO
41g/L; MgSO
46H
2O1g/L.PH5.5,121 ℃ of sterilization 20min.
Fermention medium: yeast extract 10g/L; Maltodextrin 35g/L; Sunflower seed oil 5g/L; KCl1g/L; (NH
4)
2HPO
41g/L; K
2HPO
41g/L; MgSO
46H
2O1g/L.PH5.5,121 ℃ of sterilization 20min.
Conversion process: the inclined-plane that will inoculate bacterial classification places 28 ℃ of constant incubators, cultivates 6 days, obtains sophisticated spore; Sophisticated then spore inoculating shakes in the bottle in the 250ml that the 30ml seed culture medium is housed, and on 30 ℃, 230rpm shaking table, cultivates 3 days, obtains sophisticated seed; The 750ml that the seed that obtains is equipped with the 100ml fermention medium with 10% inoculum size access shakes in the bottle; Place 30 ℃, 230r/min shaking table to continue to cultivate 20 hours, add substrate, place 28 ℃, put bottle when the 240r/min shaking table continues to be converted into 48 hours with the amount of every liter of fermented liquid 8g substrate; Get fermented liquid 2ml and add 8ml methyl alcohol soaked overnight; The centrifuging and taking supernatant is analyzed with HPLC, adopts external standard method that sample is carried out quantitatively, and calculating transformation efficiency is 86.5%.The HPLC condition is a chromatographic instrument: Waters510 type high performance liquid chromatograph; Chromatographic column: C18 post; Detector: Waters996Photodiode Array Detector; Moving phase: methyl alcohol: water=50:50; Flow velocity: 0.8ml/min; Sample size: 5 μ l; Column temperature: room temperature.
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: peptone 20g/L; Maltodextrin 30g/L; Lard 5g/L; KCl1g/L; (NH
4)
2HPO
41g/L; K
2HPO
41g/L; MgSO
46H
2O1g/L.PH5.5,121 ℃ of sterilization 20min.
Conversion process is with embodiment 1, and last transformation efficiency is 85.1%.
Embodiment 3
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: Carnis Bovis seu Bubali cream 30g/L; Dextrin 20g/L; Yelkin TTS 1g/L; KCl1g/L; (NH
4)
2HPO
41g/L; K
2HPO
41g/L; MgSO
46H
2O1g/L.PH5.5,121 ℃ of sterilization 20min.
Conversion process is with embodiment 1, and last transformation efficiency is 85.3%.
Embodiment 4
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: yeast extract 6g/L; Semen Maydis powder 50g/L; Yelkin TTS 2g/L; KCl1g/L; (NH
4)
2HPO
41g/L; K
2HPO
41g/L; MgSO
46H
2O1g/L.PH5.5,121 ℃ of sterilization 20min.
Conversion process is with embodiment 1, and last transformation efficiency is 84.3%.
Embodiment 5
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: yeast extract 12g/L; Maltodextrin 35g/L; Soya-bean oil 0.5g/L; KCl1g/L; (NH
4)
2HPO
41g/L; K
2HPO
41g/L; MgSO
46H
2O1g/L.PH5.5,121 ℃ of sterilization 20min.
Conversion process is with embodiment 1, and last transformation efficiency is 84.1%.
Embodiment 6
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: yeast extract 12g/L; Zulkovsky starch 35g/L; Soya-bean oil 5g/L; KCl1g/L; (NH
4)
2HPO
41g/L; K
2HPO
41g/L; MgSO
46H
2O1g/L.PH5.5,121 ℃ of sterilization 20min.
Conversion process is with embodiment 1, and last transformation efficiency is 86.1%.
Embodiment 7
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: yeast extract 12g/L; Maltodextrin 35g/L; Soya-bean oil 10g/L; KCl1g/L; (NH
4)
2HPO
41g/L; K
2HPO
41g/L; MgSO
46H
2O1g/L.PH5.5,121 ℃ of sterilization 20min.
Conversion process is with embodiment 1, and last transformation efficiency is 87.1%.
Embodiment 8
7.5L fermentor tank transforms synthetic I
Each substratum is with embodiment 4
Conversion process: the inclined-plane that will inoculate bacterial classification places 28 ℃ of constant incubators, cultivates 6 days, obtains sophisticated spore; Sophisticated then spore inoculating shakes in the bottle in the 750ml that the 100ml seed culture medium is housed, and on 30 ℃, 230rpm shaking table, cultivates 3 days, obtains sophisticated seed; The seed that obtains inserts with 10% inoculum size in the fermention medium of 4.5L (being loaded in the 7.5L fermentor tank); 30 ℃ of temperature, air flow 0.4VVM, rotating speed 180rpm cultivates.Cultivate after 20 hours, add substrate with the amount of every liter of fermented liquid 8g substrate, temperature transfers to 28 ℃ and begins to transform then, in conversion process, controls dissolved oxygen through increasing rotating speed more than 20% saturation ratio with air flow.Confirm transforming degree with thin layer chromatography in the conversion process, analytical procedure: take a morsel conversion fluid in centrifuge tube, add equal-volume ETHYLE ACETATE; Behind ultrasonic cell-break in 4000r/min, 25 ℃ of centrifugal 6min; Check plate on taking a morsel, developping agent is: trichloromethane: absolute ethyl alcohol=10:1 (V/V), spray the post-heating colour developing with 10% sulphuric acid soln; Observe the spot situation, confirm transforming degree.Product stops accumulation during by 48 hours, finishes to transform to put jar, and it is 85.4% that HPLC analyzes transformation efficiency.
Claims (3)
1. one kind is bacterial classification with flax thorn dish spore AS 3.4486; Biosynthesizing 7 α, the fermention medium of 15 alpha-dihydroxy-dihydrotestosterones comprises organic nitrogen source, carbon source, inorganic salt, metals ion and natural fats and oils; It is characterized in that described organic nitrogen source is a yeast extract;
The content of described organic nitrogen source is 10g/L～15g/L;
Described carbon source is the polyose carbon source, and said polyose carbon source is dextrin, starch or Semen Maydis powder; Its content is 20g/L～50g/L;
Said natural fats and oils is soya-bean oil, rapeseed oil, corn embryo oil, sunflower seed oil, Oleum Gossypii semen, lard, sheep oil, butter, fish oil, Yelkin TTS, POPG or phosphatidic acid;
The content of said natural fats and oils is 0.5g/L～10g/L.
2. fermention medium as claimed in claim 1 is characterized in that, said polyose carbon source is for being maltodextrin or Zulkovsky starch, and its content is 35g/L.
3. fermention medium as claimed in claim 1 is characterized in that, the content of said natural fats and oils is 2g/L～5g/L.
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| CN102477454B (en) * | 2010-11-30 | 2014-11-05 | 上海来益生物药物研究开发中心有限责任公司 | Preparation method for pyrone compound |
| CN102599433B (en) * | 2012-04-05 | 2013-08-07 | 安徽燕之坊食品有限公司 | Production method for coarse-cereal artificial rice |
| CN102757998A (en) * | 2012-05-28 | 2012-10-31 | 江南大学 | Method for preparing 7alpha, 15alpha-dihydroxy androstenone by flowingly addition of hydrogen peroxide |
| CN102757997A (en) * | 2012-05-28 | 2012-10-31 | 江南大学 | Method for preparing 7alpha, 15alpha-dihydroxy androstenone by pre-inducing substrate |
| CN103060201A (en) * | 2012-11-01 | 2013-04-24 | 江南大学 | Strain capable of efficiently converting DHEA (dehydroepiandrosterone) into 3 beta, 7 alpha and 15 alpha-trihydroxy androstane-5-alkene-17-ketone obtained by compound mutation screening |
| CN104673677A (en) * | 2014-04-08 | 2015-06-03 | 丽江映华生物药业有限公司 | Colletotrichum lini strain LJYH20130405-1 for steride fermentation as well as application technology thereof |
| CN104004802A (en) * | 2014-06-03 | 2014-08-27 | 济南汇隆生物科技有限公司 | Cottonseed fine meal for fermentation and preparation method thereof |
| CN106906250A (en) * | 2017-04-18 | 2017-06-30 | 四川龙蟒福生科技有限责任公司 | A kind of fermentation process for producing S abscisic acids |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002218970A (en) * | 2001-01-24 | 2002-08-06 | Kikkoman Corp | Method for producing liquid koji |
| WO2005030976A2 (en) * | 2003-09-26 | 2005-04-07 | Biosynergy Gmbh | Method for producing a carotinoid-containing biomass |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002218970A (en) * | 2001-01-24 | 2002-08-06 | Kikkoman Corp | Method for producing liquid koji |
| WO2005030976A2 (en) * | 2003-09-26 | 2005-04-07 | Biosynergy Gmbh | Method for producing a carotinoid-containing biomass |
Non-Patent Citations (2)
| Title |
|---|
| I. taxonomy et al.Tiacumicins, a novel complex of 18-membered macrolide antibiotics.<The journal of antibiotics>.1987,第40卷(第5期), * |
| I.taxonomyetal.Tiacumicins a novel complex of 18-membered macrolide antibiotics.<The journal of antibiotics>.1987 |
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