CN102703558B - Method for hydroxylating dehydroisoandrosterone by using colletotrichumlini - Google Patents
Method for hydroxylating dehydroisoandrosterone by using colletotrichumlini Download PDFInfo
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- CN102703558B CN102703558B CN201210167406.XA CN201210167406A CN102703558B CN 102703558 B CN102703558 B CN 102703558B CN 201210167406 A CN201210167406 A CN 201210167406A CN 102703558 B CN102703558 B CN 102703558B
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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 the method for 7 Alpha-hydroxies-dehydroepiandros-sterone (7 α-OH-DHEA) and 7,15 Alpha-hydroxies-dehydroepiandros-sterone (7,15 α-OH-DHEA).
Background technology
7 Alpha-hydroxies-dehydroepiandros-sterone (7 α-OH-DHEA) and 7,15 Alpha-hydroxies-dehydroepiandros-sterone (7,15 α-OH-DHEA) are important active substances in organism, and metabolism is had to important regulating effect.At present, there are some researches show that 7 α-OH-DHEA can stop the hypoxic cell of extracorporeal neuron dead, and there is antioxygenation and antitumor action.7,15 Alpha-hydroxies-dehydroepiandros-sterone (7,15 α-OH-DHEA), is a kind of important Steroid medicine intermediates, can be used for synthetic multiple steroid hormone class medicine, as the principal constituent of synthetic " the 4th generation " oral contraceptive is bent sieve ketone.Because above-mentioned two kinds of hydroxy-steroids all have valuable pharmacological effect and pharmaceutical use, its market outlook are considerable, become a large focus of current steroid drugs area research.
From nineteen fifty-two, the Murray of Upjohn company and Peterson application
rhizopus nigricansthe C11 of Progesterone is introduced 11 α-OH in success, has solved the hang-up that cortisone is produced, and from then on microorganism Steroid Transformation technology has caused people's great attention.In recent years, investigator starts to utilize biotransformation method to import hydroxyl in the C7 position of DHEA both at home and abroad, but relatively less about the research of DHEA dihydroxylation.In steroidal microbial transformation process, water-soluble low, the biocompatibility of substrate is poor is the key factor of restriction conversion rate, directly becomes the bottleneck in microbial transformation steroidal compounds industrial production.For this present situation, many investigators start to improve the feeding mode of substrate, as ultrasonic wave refinement, organic solvent hydrotropy and double water-phase catalysis etc., but because organic solvent has certain toxicity to microorganism conventionally, can cause restraining effect to a certain degree to enzymic activity, therefore little to the increase rate of transformation efficiency.Therefore, adopt a kind of efficient substrate feeding mode to have very important significance, and at present about first group-beta-cyclodextrin (M-β-CD) for flax thorn dish spore mould (
colletotrichum lini) research of hydroxylation DHEA has no report, its water-soluble and transformation efficiency that significantly improves substrate is industrial one large advantage especially.
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, by adding appropriate first group-beta-cyclodextrin (M-β-CD), reach the feed intake requirement of concentration, high substrate high conversion, high efficiency of pcr product of high substrate.This bacterial classification is preserved in China Committee for Culture Collection of Microorganisms's 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, and Classification And Nomenclature is flax thorn dish spore mould (Colletotrichum lini).
Application aforesaid method transforms the method that DHEA produces 7 α-OH-DHEA and 7,15 α-OH-DHEA, and its step is as follows:
(1) prepare the inclusion compound of DHEA/M-β-CD: take DHEA, the M-β-CD that mol ratio is 1:1 and be dissolved in suitable quantity of water, stir at 30 ℃ after 24 h by the film of 0.45 μ m, filtrate lyophilize obtains 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 ℃, under 120-220 r/min condition, activation culture obtains seed liquor, then by seed liquor dilution spread to 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 250 mL Erlenmeyer flasks of the sterilized 20-50 of being equipped with mL seed culture medium, culture temperature is 25-40 ℃, puts on shaking table with the rotating speed of 120-220 r/min and cultivates 12-16 h to logarithmic growth mid-term, obtains
c.linithe cell liquid culture of ST-1 bacterial strain.
(4) fermentation culture: by the cell liquid culture preparing in step (2) with 8-10%(v/v) inoculum size access fermention medium, liquid amount is in 250 mL Erlenmeyer flasks, to fill 20-50 mL fermention medium, culture temperature is 25-40 ℃, under the rotating speed of 120-220 r/min, cultivate 20-24 h, obtain fermented liquid.
(5) bio-transformation: the inclusion compound that takes the DHEA/M-β-CD described in appropriate step (1), drop into the thalline fermented liquid in step (3), making its final concentration is 18-20 g/L, 28 ℃ of invert points, under the rotating speed of 200-220 r/min, transform 48-60 h, obtain conversion fluid.
(6) product detects: by the conversion fluid of step (4) centrifugal 5-10 min under 8000-12000 r/min, equal-volume ethyl acetate extracting 3 times for supernatant, appropriate chloroform extracting 3 times for thalline, after merging extract, in Rotary Evaporators, be threaded to crystal appearance, acetonitrile redissolves crystal also by the organic membrane filter removal of impurities of 0.22 μ m, filtrate 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 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; PH nature, sterilizing 20min under 121 ℃ of high pressure steam; Composition and the proportioning of the described 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; Before sterilizing, adjust the pH to 6.5-7.5 of substratum, sterilizing 20min under 121 ℃ of high pressure steam;
Composition and the proportioning of the described 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, sterilizing 20 min under 121 ℃ of high pressure steam.
Of the present invention utilize flax thorn dish spore mould (
colletotrichum lini) method of ST-1 hydroxylation dehydroepiandros-sterone, to propose first first substrate DHEA and appropriate M-β-CD to be carried out to the transform mode feeding intake again after pre-inclusion processing, finally reach the feed intake object of concentration, high substrate conversion efficiency, high efficiency of pcr product of high substrate, this method has great importance to microbial transformation steroid compound.
Accompanying drawing explanation
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 is prepared inclusion compound DHEA/M-β-CD.
(1) phase equilibrium line is determined inclusion ratio.
Take appropriate M-β-CD, in 250 mL triangular flasks, fill 30mL deionized water and be configured to the concentration gradient that concentration is 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, add respectively excessive substrate DHEA to be placed on 20-40 ℃, 24 h that vibrate on the shaking table of 120-200 r/min are to inclusion balance.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, recycles high performance liquid chromatography detection substrate concentration after suitable multiple dilution.M-β-CD concentration of take is X-coordinate, and DHEA concentration is that ordinate zou is drawn phase equilibrium line, determines the inclusion ratio of DHEA and M-β-CD.
(2) lyophilization is prepared the inclusion compound of DHEA/M-β-CD.
Take DHEA, the M-β-CD that mol ratio is 1:1 and be dissolved in suitable quantity of water, stir at 20-40 ℃ 24 h to after inclusion process balance with the water film filtering removal of impurities of 0.45 μ m, after filtrate lyophilize, obtain the inclusion compound of DHEA/M-β-CD.
(1) prepare flax thorn dish spore mould (
colletotrichum lini) the cell liquid culture of ST-1 bacterial strain
Flax thorn dish spore on 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, at 30 ℃, be placed on shaking table and cultivate 20-24 h to logarithmic phase with the rotating speed of 200 r/min, 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; Corn steep liquor 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, sterilizing 20 min under 121 ℃ of high pressure steam.
(4) shake flask fermentation: by above-mentioned flax thorn dish spore mould (
colletotrichum lini) the cell liquid culture of ST-1 bacterial strain is with 8-10%(w/w) and inoculum size be seeded in and be equipped with in the sterilized 250 mL Erlenmeyer flasks that 30 mL fermention mediums are housed, under 20-40 ℃ of condition, being placed in the rotating speed with 120-200 r/min on shaking table cultivates, fermentation 20-24 h, thalline enters logarithmic phase.
(5) bio-transformation: accurately take appropriate DHEA/M-beta-CD inclusion, drop into the thalline fermented liquid in step (3), making DHEA final concentration is 20 g/L, 28 ℃ of invert points, transforms 48-60 h under the rotating speed of 200-220 r/min.
Claims (2)
1. a method of utilizing flax thorn dish spore mould ST-1 hydroxylation dehydroepiandros-sterone (DHEA), this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center that is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on April 24th, 2012, deposit number is CGMCC No.6051, called after flax thorn dish spore of this bacterial strain mould (
colletotrichum lini), it is characterized by: the fermention medium of the bottled 20-40 mL of 250 mL triangle, inoculum size is 8-10%(w/w), culture temperature 25-40 ℃, shaking speed 180-220 r/min, after cultivation 20-24 h, drop into the inclusion compound of appropriate DHEA/ first group-beta-cyclodextrin (M-β-CD), 28 ℃ of invert points, continue to transform 48-60 h.
Flax thorn dish spore as claimed in claim 1 mould (
colletotrichum lini) method of ST-1 hydroxylation DHEA, it is characterized in that, fermention medium used is composed 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, sterilizing 20 min under 121 ℃ of high pressure steam.
Flax thorn dish spore as claimed in claim 1 mould (
colletotrichum lini) method of ST-1 hydroxylation DHEA, it is characterized in that, the preparation method of described inclusion compound is as follows: DHEA and M-β-CD are dissolved in suitable quantity of water with mol ratio 1:1, stirs at 30 ℃ after 24 h by the film of 0.45 μ m, filtrate lyophilize.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Families Citing this family (3)
| 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 |
| 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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| 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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| 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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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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