CN102703558A - Method for hydroxylating dehydroisoandrosterone by using colletotrichumlini - Google Patents
Method for hydroxylating dehydroisoandrosterone by using colletotrichumlini Download PDFInfo
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- CN102703558A CN102703558A CN201210167406XA CN201210167406A CN102703558A CN 102703558 A CN102703558 A CN 102703558A CN 201210167406X A CN201210167406X A CN 201210167406XA CN 201210167406 A CN201210167406 A CN 201210167406A CN 102703558 A CN102703558 A CN 102703558A
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- dhea
- beta
- cyclodextrin
- inclusion compound
- colletotrichumlini
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Abstract
The invention relates to a method for hydroxylating dehydroisoandrosterone (DHEA) by using colletotrichumlini ST-1. The method for hydroxylating the dehydroisoandrosterone by using the colletotrichumlini ST-1 is characterized in that: in a conversion process, a way of pre-including the DHEA and methyl-beta-cyclodextrin with an equimolar ratio first and then feeding is adopted, so that the water solubility and the conversion efficiency of the DHEA are significantly improved. Under a condition that a substrate feeding amount is 10g/L, the conversion rate is as high as 95% and the total yield of products 7 alpha-OH-dehydroepiandrosterone and 7,15 alpha-OH-dehydroepiandrosterone is 80.94%.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of efficiently utilize flax thorn dish spore mould (
Colletotrichum lini) ST-1 hydroxylation dehydroepiandros-sterone (DHEA) is 7 Alpha-hydroxies-dehydroepiandros-sterone (7 α-OH-DHEA) and 7,15 Alpha-hydroxies-dehydroepiandros-sterone (method of 7,15 α-OH-DHEA).
Background technology
((7,15 α-OH-DHEA) are active substances important in the organism to 7 Alpha-hydroxies-dehydroepiandros-sterone, and metabolism is had important regulatory role for 7 α-OH-DHEA) and 7,15 Alpha-hydroxies-dehydroepiandros-sterone.At present, there are some researches show that 7 α-OH-DHEA can stop the hypoxic cell of extracorporeal neuron dead, and have antioxygenation and antitumor action.7,15 Alpha-hydroxies-dehydroepiandros-sterone (7,15 α-OH-DHEA), be a kind of important steroid drugs midbody, can be used for synthetic multiple steroid hormone class medicine, bend sieve ketone like the principal constituent of synthetic " the 4th generation " oral contraceptive.Because above-mentioned two kinds of hydroxy-steroids all have valuable pharmacological effect and pharmaceutical use, its market outlook are considerable, become a big focus of current steroid drugs area research.
From nineteen fifty-two, the Murray of Upjohn company and Peterson use
Rhizopus nigricansSuccess has solved the hang-up that KE is produced to the C11 of 11 α-OH introducing Progesterone, and from then on mikrobe steroidal transformation technology has caused people's great attention.In recent years, the investigator begins to utilize biotransformation method to import hydroxyl in the C7 position of DHEA both at home and abroad, but less relatively about the research of DHEA dihydroxylation.In the steroidal microbial transformation process, water-soluble low, the biocompatibility difference of substrate is the key factor of restriction conversion rate, directly becomes the bottleneck in the microbial transformation steroidal compounds industrial production.To this present situation; Many investigators begin to improve the feeding mode of substrate; Like UW refinement, organic solvent hydrotropy and double water-phase catalysis etc.; But, can cause restraining effect to a certain degree to enzymic activity, so little to the increase rate of transformation efficiency because organic solvent has certain toxicity to mikrobe usually.Therefore, adopt a kind of feeding mode of substrate efficiently to have very important significance, and at present about methyl-beta-cyclodextrin (M-β-CD) be used for flax thorn dish spore mould (
Colletotrichum lini) research of hydroxylation DHEA do not appear in the newspapers, it significantly improves the water-soluble and transformation efficiency industrial especially one big advantage of substrate.
Summary of the invention
The object of the present invention is to provide a kind of efficiently utilize flax thorn dish spore mould (
Colletotrichum lini) method of ST-1 hydroxylation dehydroepiandros-sterone, (M-β-CD) reaches the feed intake requirement of concentration, high substrate high conversion, high efficiency of pcr product of high substrate through adding an amount of methyl-beta-cyclodextrin.This bacterial classification is preserved in the China Committee for Culture Collection of Microorganisms common micro-organisms center of No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on April 24th, 2012; Deposit number is CGMCC No.6051, classification called after flax thorn dish spore mould (Colletotrichum lini).
Use aforesaid method and transform the method that DHEA produces 7 α-OH-DHEA and 7,15 α-OH-DHEA, its step is following:
(1) inclusion compound of preparation DHEA/M-β-CD: take by weighing DHEA, the M-β-CD that mol ratio is 1:1 and be dissolved in the suitable quantity of water, 30 ℃ are stirred down behind 24 h through the film of 0.45 μ m, and the filtrating lyophilize promptly gets the inclusion compound of DHEA/M-β-CD.
(2) adopt preserving number be flax thorn dish spore of CGMCC No.4903 mould (
Colletotrichum lini) ST-1 is for producing bacterial classification, 25-40 ℃, activation culture obtains seed liquor under the 120-220 r/min condition, then the seed liquor dilution is applied on the PDA flat board.
(3) preparation
C.liniThe cell liquid culture of ST-1 bacterial strain: flax thorn dish spore on the PDA solid medium of picking step (1) mould (
Colletotrichum lini) ST-1 bacterial strain one ring, being inoculated in the 250 mL Erlenmeyer flasks of the sterilized 20-50 of being equipped with mL seed culture medium, culture temperature is 25-40 ℃, puts on the shaking table with the rotating speed of 120-220 r/min and cultivates 12-16 h to logarithmic growth mid-term, promptly obtains
C.liniThe cell liquid culture of ST-1 bacterial strain.
(4) fermentation culture: with the inoculum size access fermention medium of the cell liquid culture for preparing in the step (2) with 8-10% (v/v); Liquid amount is a dress 20-50 mL fermention medium in the 250 mL Erlenmeyer flasks; Culture temperature is 25-40 ℃; Under the rotating speed of 120-220 r/min, cultivate 20-24 h, get fermented liquid.
(5) bio-transformation: the inclusion compound that takes by weighing the DHEA/M-β-CD described in an amount of step (1); Drop into the thalline fermented liquid in the step (3), making its final concentration is 18-20 g/L, 28 ℃ of invert points; The rotating speed of 200-220 r/min transforms 48-60 h down, promptly gets conversion fluid.
(6) product detects: with conversion fluid centrifugal 5-10 min under 8000-12000 r/min of step (4); Supernatant is with equal-volume ETHYLE ACETATE extracting 3 times; Thalline has been threaded to crystal behind the merging extract and has occurred with an amount of chloroform extracting 3 times in Rotary Evaporators, acetonitrile redissolution crystal also passes through the organic membrane filter removal of impurities of 0.22 μ m; Filtrating is utilized the content of high-efficient liquid phase chromatogram technique analysis DHEA, 7 α-OH-DHEA and 7,15 α-OH-DHEA.
Wherein the composition of the PDA substratum described in the step (2) and proportioning are: potato 200-500 g/L; Glucose 20-50 g/L; Yeast powder 2-10 g/L; Agar powder 10-20 g/L; The pH nature, sterilization 20min under 121 ℃ of HP steams; The composition and the proportioning of the said seed culture medium of step (2) are: groundnut meal 5-15 g/L, glucose 20-50 g/L; KCl 0.1-1 g/L; Yeast powder 10-30g/L; K
2HPO
40.1-1.0 g/L; MgSO
47H
2O 0.01-0.1 g/L; FeSO
47 H
2O 0.01-0.1 g/L g/L; Transfer the pH to 6.5-7.5 of substratum before the sterilization, sterilization 20min under 121 ℃ of HP steams;
The composition and the proportioning of the said fermention medium of step (4) are: glucose 10-100 g/L; Yeast powder 5-50g/L; Peptone 5-50 g/L; NaCl 0.2-2 g/L; K
2HPO
40.1-1.0 g/L; KH
2PO
40.1-1.0 g/L; MgSO
40.01-0.1 g/L; FeSO
47 H
2O 0.01-0.10 g/L; PH 6.5-7.5, sterilization 20 min under 121 ℃ of HP steams.
Of the present invention utilize flax thorn dish spore mould (
Colletotrichum lini) method of ST-1 hydroxylation dehydroepiandros-sterone; Be to propose first earlier substrate DHEA and an amount of M-β-CD are carried out the transform mode that feeds intake again after preparatory inclusion is handled; Finally reach the feed intake purpose of concentration, high substrate conversion efficiency, high efficiency of pcr product of high substrate, this method has great importance to the microbial transformation steroid compound.
Description of drawings
Fig. 1 DHEA/M-β-CD inclusion compound phase equilibrium line.
Fig. 2 be flax thorn dish spore of the present invention mould (
Colletotrichum lini) ST-1 transforms the process study of DHEA/M-beta-CD inclusion in fermention medium.
Embodiment
Embodiment 1 lyophilization prepares inclusion compound DHEA/M-β-CD.
(1) phase equilibrium line is confirmed the inclusion ratio.
Take by weighing an amount of M-β-CD; Dress 30mL deionized water is configured to the concentration gradient that concentration is 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM in the 250 mL triangular flasks; Add excessive substrate DHEA respectively and be placed on 20-40 ℃, vibration 24 h are to the inclusion balance on the shaking table of 120-200 r/min.Centrifugal 3 min of inclusion liquid 1000 r remove the not substrate of inclusion, and supernatant with the water film filtering removal of impurities of 0.22 μ m, utilizes HPLC to detect concentration of substrate after the suitable multiple dilution again.With M-β-CD concentration is X-coordinate, and DHEA concentration is that ordinate zou is drawn phase equilibrium line, confirms the inclusion ratio of DHEA and M-β-CD.
(2) lyophilization prepares the inclusion compound of DHEA/M-β-CD.
Take by weighing DHEA, the M-β-CD that mol ratio is 1:1 and be dissolved in the suitable quantity of water, 20-40 ℃ is stirred down 24 h water film filtering removal of impurities with 0.45 μ m to the inclusion process balance, promptly gets the inclusion compound of DHEA/M-β-CD after the lyophilize of filtrating.
(1) preparation flax thorn dish spore mould (
Colletotrichum lini) the cell liquid culture of ST-1 bacterial strain
Flax thorn dish spore on the picking solid PDA substratum mould (
Colletotrichum lini) ST-1 bacterial strain one ring, be seeded in the 250 mL Erlenmeyer flasks that 30 mL seed culture mediums are housed, under 30 ℃, place on the shaking table and cultivate 20-24 h to logarithmic phase with the rotating speed of 200 r/min, promptly make the cell liquid culture of flax thorn dish spore trichoderma strain.
(3) composition of fermention medium and proportioning are: glucose 20-50 g/L; Yeast powder 10-30g/L; Peptone 5-20 g/L; Steeping water 3-10 g/L; NaCl 0.2-2 g/L; K
2HPO
40.1-1.0 g/L; MgSO
40.1-1 g/L; FeSO
47 H
2O 0.01-0.10 g/L; PH 6.5-7.5, sterilization 20 min under 121 ℃ of HP steams.
(4) shake flask fermentation: with above-mentioned flax thorn dish spore mould (
Colletotrichum lini) the cell liquid culture of ST-1 bacterial strain is seeded in the inoculum size of 8-10% (w/w) and is equipped with in the sterilized 250 mL Erlenmeyer flasks that 30 mL fermention mediums are housed; Under 20-40 ℃ of condition; Place on the shaking table with the rotating speed of 120-200 r/min and cultivate; Fermentation 20-24 h, thalline gets into logarithmic phase.
(5) bio-transformation: accurately take by weighing an amount of DHEA/M-beta-CD inclusion, drop into the thalline fermented liquid in the step (3), making the DHEA final concentration is 20 g/L, and 28 ℃ of invert points, the rotating speed of 200-220 r/min transforms 48-60 h down.
Claims (3)
- One kind novel utilize flax thorn dish spore mould ( Colletotrichum lini) method of ST-1 hydroxylation dehydroepiandros-sterone (DHEA); It is characterized by: the fermention medium of the bottled 20-40 mL of 250 mL triangles; Inoculum size is 8-10% (w/w), culture temperature 25-40 ℃, and shaking speed 180-220 r/min; Cultivate and to drop into an amount of DHEA/ methyl-beta-cyclodextrin behind the 20-24 h (inclusion compound of M-β-CD) continues to transform 48-60 h.
- 2. the method for claim 1 is characterized in that, used fermention medium is formed as follows: glucose 10-100 g/L; Yeast powder 5-50g/L; Peptone 5-50 g/L; NaCl 0.2-2 g/L; K 2HPO 40.1-1.0 g/L; KH 2PO 40.1-1.0 g/L; MgSO 40.01-0.1 g/L; FeSO 47 H 2O 0.01-0.10 g/L; PH 6.5-7.5, sterilization 20 min under 121 ℃ of HP steams.
- 3. the method for claim 1; It is characterized in that; (preparation method of the inclusion compound of M-β-CD) is following: DHEA and M-β-CD are dissolved in the suitable quantity of water with mol ratio 1:1 said DHEA/ methyl-beta-cyclodextrin, 30 ℃ of films that stir down behind 24 h through 0.45 μ m, filtrating lyophilize.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103911418A (en) * | 2014-03-14 | 2014-07-09 | 江南大学 | A method of efficiently catalyzing dehydroepiandrosterone by utilization of a resting cell of colletotrichum linli |
| CN104561216A (en) * | 2014-11-09 | 2015-04-29 | 浙江仙居君业药业有限公司 | Method for preparing 7alpha,15alpha-dyhydroxyldehydroepiandrosterone by utilizing flax colletotrichum mould |
| CN104611400A (en) * | 2014-11-25 | 2015-05-13 | 江南大学 | Method of utilizing coenzyme regeneration and resin in-situ extraction to promote hydroxylation of DHEA by Colletotrichum lini ST-1 |
Citations (1)
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| CN101182565A (en) * | 2006-11-14 | 2008-05-21 | 上海医药工业研究院 | Extraction and purification method of 7 Alpha, 15Alpha-dihydroxy androstene alcohol ketone |
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2012
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Patent Citations (1)
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| CN101182565A (en) * | 2006-11-14 | 2008-05-21 | 上海医药工业研究院 | Extraction and purification method of 7 Alpha, 15Alpha-dihydroxy androstene alcohol ketone |
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| 《Microbiology》 20071231 M.V.Donova Methyl-beta-cyclodextrin alters growth, actibity and cell enbelope features of sterol-transforming mycobacteria 1981-1992 1-3 第153卷, * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103911418A (en) * | 2014-03-14 | 2014-07-09 | 江南大学 | A method of efficiently catalyzing dehydroepiandrosterone by utilization of a resting cell of colletotrichum linli |
| CN104561216A (en) * | 2014-11-09 | 2015-04-29 | 浙江仙居君业药业有限公司 | Method for preparing 7alpha,15alpha-dyhydroxyldehydroepiandrosterone by utilizing flax colletotrichum mould |
| CN104561216B (en) * | 2014-11-09 | 2019-01-11 | 浙江仙居君业药业有限公司 | It is a kind of to pierce 7 α of the mould preparation of disk spore, the method for 15 alpha-dihydroxy dehydroepiandros-sterones using flax |
| CN104611400A (en) * | 2014-11-25 | 2015-05-13 | 江南大学 | Method of utilizing coenzyme regeneration and resin in-situ extraction to promote hydroxylation of DHEA by Colletotrichum lini ST-1 |
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| CN102703558B (en) | 2014-01-15 |
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Effective date of registration: 20190312 Address after: 317300 Lingxiu Road, Yangfu Industrial Park, Xianju County, Taizhou City, Zhejiang Province Co-patentee after: Jiangxi Junye Biopharmaceutical Co., Ltd. Patentee after: Zhejiang Xianju Junye Pharmaceutical Co., Ltd. Address before: 317300 Junye Road, Xianju County, Taizhou City, Zhejiang Province Patentee before: Zhejiang Xianju Junye Pharmaceutical Co., Ltd. |