CN105543320B - A method of promote reducing power regeneration to improve microorganism and is hydroxylated DHEA transformation efficiency - Google Patents
A method of promote reducing power regeneration to improve microorganism and is hydroxylated DHEA transformation efficiency Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of 5L fermentor technique of flax thorn mould (Colletotrichum lini) ST-1 Efficient Conversion DHEA of disk spore.Technical solution of the present invention includes actication of culture, seed culture, DHEA bioconversion, product detection, by the monitoring to the indexs such as base consumption, residual sugar content in conversion process, substrate will be added in batches with promoting reducing power regeneration to combine and solves the problems, such as that substrate feed concentrations and transformation efficiency are all relatively low.The method improves efficiency of pcr product, shortens the transformation period, easy to operate, and inventory is high, the large-scale production suitable for steroidal compounds bioconversion.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of high concentration dehydroepiandros-sterone (DHEA) promotees under the conditions of feeding intake
It is 7 α, 15 Alpha-hydroxies-that mould (Colletotrichum lini) the ST-1 hydroxylation DHEA of flax thorn disk spore is improved into reducing power regeneration
The method of dehydroepiandros-sterone (7 α, 15 α-diOH-DHEA).
Background technique
Dehydroepiandros-sterone (DHEA) is by a kind of precursor of adrenal hormone Sulfuric acid DHEA (DHEAS) of acth secretion
Matter, treat the nervous system disease and in terms of have extensive pharmacological activity.DHEA can be used as intermediate
The many steroidal compounds with important physiological activity of synthesis, such as 7 α, 15 α-diOH-DHEA are synthesizing new oral contraceptives
The precursor compound of the effective component Drospirenone of " excellent think of is bright ".Due to 7 α, 15 α-diOH-DHEA have important pharmacological action and
Medical value, market prospects are very considerable, therefore how to improve substrate DHEA feed concentrations and 7 α, and 15 α-diOH-DHEA are produced
Object yield becomes a big hot spot of current steroid drugs area research.
Currently, from nature screening obtain it is multiple have hydroxylation DHEA generate 7 α, the bacterium of 15 α-diOH-DHEA abilities
Strain.Wherein, Colletotrichum lini is most strong to DHEA hydroxylation ability, and under shake flask culture conditions, substrate feed concentrations are
When 10g/L, 7 α, 15 α-diOH-DHEA yield can reach 30% or so.But since bacterial strain activity of conversion is not high, high concentration substrate
The essential problem for waiting thallus toxic action bioconversions by force, causes 7 α of substrate feed concentrations and product in conversion process, 15 α-
DiOH-DHEA yield is all relatively low, and these problems are directly becoming 7 α, the bottleneck in 15 α-diOH-DHEA industrial productions.Steroidal
Hydroxylation reaction is the Monooxygenase activity for the Cytochrome P450 being utilized on eukaryocyte mitochondrial membrane, the reaction of hydroxylation reaction
Formula is as follows:
Substrate+NADPH+O2→ product+NADP++H2O
By the reaction equation it is found that the offer of substrate feed concentrations, reducing power is the important of influence steroidal hydroxylation reaction efficiency
Factor.In steroidal dihydroxylation process, NADPH is constantly consumed, and enough reducing powers cannot be provided for hydroxylation reaction, thus direct shadow
Ring being normally carried out for hydroxylation reaction.External source adds glucide, promotes reducing power NADPH again using microorganism own metabolism approach
Raw, simple process is economic and practical.In order to solve toxic action of the high concentration substrate to thallus, 7 α of product, 15 α-diOH- are improved
DHEA yield, this patent establishes one kind and adds Binding Capacity promotion reducing power regeneration in batches, suitable for large-scale production
The 5L fermentor conversion process of C.lini ST-1 conversion DHEA.
Summary of the invention
Promote reducing power regeneration to improve under the conditions of feeding intake the purpose of the present invention is to provide a kind of high concentration DHEA
C.lini ST-1 is hydroxylated DHEA and generates 7 α, the method for 15 α-diOH-DHEA.The production bacterial strain that the present invention uses is in April, 2012
It is preserved within 24th the Chinese microorganism strain of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
It is mould that flax that preservation administration committee common micro-organisms center deposit number is CGMCC No.6501 pierces disk spore
(Colletotrichum lini) ST-1 reduces toxic action of the high concentration substrate to thallus by the way that substrate DHEA is added portionwise,
Add suitable glucose simultaneously enough nutriments be provided and promote reducing power regeneration, finally realize high substrate feed concentrations,
The requirement of high substrate high conversion, high efficiency of pcr product.
7 α are produced using mentioned microorganism conversion DHEA, the method for 15 α-diOH-DHEA, its step are as follows:
(1) use deposit number for CGMCC No.6501 flax pierce mould (Colletotrichum lini) ST-1 of disk spore be
Strain is produced, 25-40 DEG C, activation culture obtains seed liquor under the conditions of 120-220r/min, which is applied to PDA culture
On base；
(2) the cellular liquid culture of C.lini ST-1 bacterial strain is prepared: on the PDA solid medium of picking step (1)
One ring of C.lini ST-1 bacterial strain is inoculated in the sterilized 250mL conical flask equipped with 20-50mL seed culture medium, culture temperature
Degree is 25-40 DEG C, is set on shaking table with the revolving speed culture 18-30h of 120-220r/min to mid log phase to get to C.lini
The cellular liquid culture of ST-1 bacterial strain；
(3) thalli growth in 5L fermentor: by the cellular liquid culture prepared in step (2) with 8-10% (v/v)
Inoculum concentration access equipped with fermentation medium 5L fermentor in, liquid amount 3L.In growth phase, cultivation temperature 25-40
DEG C, tank pressure is set as 0.05MPa, and revolving speed is set as 220-300r/min, ventilatory capacity 0.4-1.0vvm.By adjusting the logical of fermentor
Tolerance, revolving speed and tank pressure are maintained at 10-20% to control dissolved oxygen level in fermentation liquid, grow after 12h to get to being suitable for DHEA
The liquid medium of conversion；
(4) substrate adds technique in batches: accurately weighing three parts of powdered DHEA of 5g/L, puts into respectively in 12h, 16h, 20h
Bacterial culture fluid in step (3) makes substrate DHEA feed intake final concentration of 15g/L, to reduce the toxic action of high concentration substrate.
(5) promote reducing power regeneration technology: when conversion 18h, 15g/L glucose is added as the regenerated nutrients of reducing power
Matter, to promote the regeneration of reducing power in fermentation liquid.
(6) DHEA bioconversion: in the conversion process, cultivation temperature is 25-40 DEG C, and tank pressure is set as 0.05MPa, ventilatory capacity
For 0.8-1.2vvm, revolving speed 300-450r/min, fermentation liquid is controlled by adjusting ventilatory capacity, revolving speed and the tank pressure of fermentor
Middle dissolved oxygen level is maintained at 20-30%.It maintains to turn by the sodium hydroxide of the acetic acid of fermentor auto-feeding 40% or 10%
The pH of change system is 6.5, converts 30-48h to get conversion fluid.
(7) conversion fluid of step (5) product detection: is centrifuged 5-10min, supernatant use etc. at 8000-12000r/min
Volume of ethylacetate extracts 7 times, precipitates with appropriate chloroform 3 times, has been threaded to crystalline substance in Rotary Evaporators after merging extract
Body occurs, and acetonitrile redissolves crystal and cleaned by 0.22 μm of organic membrane filter, and filtrate utilizes 7 α of high-efficient liquid phase chromatogram technique analysis,
The content of 15 α-diOH-DHEA.
The wherein ingredient and proportion of PDA culture medium described in step (1) are as follows: potato 200-500g/L；Glucose 20-
50g/L；Yeast powder 2-10g/L；Agar powder 10-20g/L；PH is naturally, the 20min that sterilizes under 121 DEG C of high steams；Step (2)
The ingredient and proportion of the seed culture medium are as follows: groundnut meal 5-15g/L, glucose 20-50g/L；KCl 1-5g/L；Yeast powder
10-30g/L；K2HPO40.1-5.0g/L；MgSO4·7H2O 0.1-5g/L；FeSO4·7H2O g/L；Culture medium is adjusted before sterilizing
PH to 6.5-7.5, sterilize 20min under 121 DEG C of high steams；The ingredient and proportion of step (3) described fermentation medium are as follows:
Glucose 20-50g/L；Yeast powder 10-30g/L；Corn pulp 3-10g/L；Sterilize 20min under 121 DEG C of high steams.Step (4)
It is described to add dextrose components and proportion are as follows: glucose 15g/L, sterilize under 121 DEG C of high steams 20min.
It is of the present invention to convert DHEA using mould (Colletotrichum lini) ST-1 of flax thorn disk spore as 7 α, 15 α-
The 5L fermentor technical study of diOH-DHEA, for the first time improves substrate feed concentrations to 15g/L, and can reach high substrate and turn
The requirement of rate, high efficiency of pcr product, the method have microorganism conversion steroidal compounds synthesizing steroid pharmaceutical intermediate important
Reference.
Detailed description of the invention
Hydroxylation of flax thorn mould (Colletotrichum lini) ST-1 of disk spore to DHEA under the conditions of Fig. 1 high substrate feeds intake
Process study.
Fig. 2 substrate adds the research that technique influences the dihydroxylation process of DHEA in batches.Different substrates are fed in batches mode such as
Under: A) accurately weigh three parts of powdered DHEA of 5g/L puts into bacterial culture fluid respectively at 12,16,20h respectively, makes substrate DHEA
Feed intake final concentration of 15g/L.B the powdered DHEA of 5g/L accurately) is weighed, bacterial culture fluid is put into 12h, puts into 10g/L after 8h
Substrate makes substrate DHEA feed intake final concentration of 15g/L.C the powdered DHEA of 10g/L accurately) is weighed, puts into thallus culture in 12h
Liquid puts into 5g/L substrate after 8h, and substrate DHEA is made to feed intake final concentration of 15g/L.
Fig. 3 substrate, which is fed in batches, influences DHEA dihydroxylation process
Research.The hydroxylation strategy is as follows: accurately three parts of powdered DHEA of 5g/L is weighed, in the thallus that 12h, 16h, 20h are put into respectively
Culture solution makes substrate DHEA feed intake final concentration of 15g/L.When converting 18h, 15g/L glucose is added.
Specific embodiment
Flax pierces mould (Colletotrichum lini) ST-1 of disk spore to DHEA's under the conditions of the high substrate of embodiment 1 feeds intake
The research of hydroxylation reaction
(1) C.lini ST-1 cellular liquid culture is prepared
C.lini ST-1 on picking PDA solid medium is inoculated in equipped with 30mL fresh liquid seed culture medium
In 250mL conical flask, 30 DEG C, 120-220r/min culture for 24 hours.Again with the inoculum concentration access of 8-10% (v/v) equipped with fermentation training
In the 5L fermentor for supporting base, liquid amount 3L.In growth phase, cultivation temperature is 25-40 DEG C, and tank pressure is set as 0.05MPa, revolving speed
It is set as 220-300r/min, ventilatory capacity 0.4-1.0vvm, dissolved oxygen level is maintained at 10-20%, grows after 12h to get to suitable
The liquid medium for being suitable for DHEA to convert；
(2) DHEA hydroxylation reaction
The powdered DHEA of 15g/L accurately is weighed, the bacterial culture fluid in 12h investment step (1).In dihydroxylation process,
Cultivation temperature is 25-40 DEG C, and tank pressure is set as 0.05MPa, pH 6.5, ventilatory capacity 0.8-1.2vvm, revolving speed 300-450r/
Min, ventilatory capacity, revolving speed and tank pressure by adjusting fermentor are maintained at 20-30% to control dissolved oxygen level in fermentation liquid.Conversion
30-48h is to get conversion fluid.
(3) product detection
Conversion fluid is collected, is extracted 7 times through ethyl acetate, crystal has been threaded in Rotary Evaporators after merging extract and has gone out
Existing, acetonitrile redissolves crystal and is cleaned by 0.22 μm of organic membrane filter, and filtrate detects 7 α, 15 α-using high performance liquid chromatography
DiOH-DHEA yield.
(4) conversion results
In 5L fermentor technical study, substrate DHEA feed concentrations are improved to 15g/L, after converting 52h, efficiency of pcr product
Only 49.1%, dry cell weight peak is only 19.1g/L.
2 substrate of embodiment adds research of the technique to the hydroxylation reaction of DHEA in batches
(1) C.lini ST-1 cellular liquid culture is prepared
C.lini ST-1 on picking PDA solid medium is inoculated in equipped with 30mL fresh liquid seed culture medium
In 250mL conical flask, 30 DEG C, 120-220r/min culture for 24 hours.Again with the inoculum concentration access of 8-10% (v/v) equipped with fermentation training
In the 5L fermentor for supporting base, liquid amount 3L.In growth phase, cultivation temperature is 25-40 DEG C, and tank pressure is set as 0.05MPa, revolving speed
It is set as 220-300r/min, ventilatory capacity 0.4-1.0vvm, dissolved oxygen level is maintained at 10-20%, grows after 12h to get to suitable
The liquid medium for being suitable for DHEA to convert；
(2) substrate is fed in batches technique
It is as follows that three kinds of different substrates are fed in batches modes: three parts of powdered DHEA of 5g/L accurately A) are weighed, respectively at 12,
16,20h puts into the bacterial culture fluid in step (1) respectively, substrate DHEA is made to feed intake final concentration of 15g/L.B it) accurately weighs
The powdered DHEA of 5g/L puts into the bacterial culture fluid in step (1) in 12h, puts into 10g/L substrate after 8h, throw substrate DHEA
Expect final concentration of 15g/L.C the powdered DHEA of 10g/L accurately) is weighed, the bacterial culture fluid in 12h investment step (1), after 8h
5g/L substrate is put into, substrate DHEA is made to feed intake final concentration of 15g/L.Under three kinds of different feeding modes, dihydroxylation process conditional
It is all the same.Cultivation temperature is 25-40 DEG C, and tank pressure is set as 0.05MPa, pH 6.5, ventilatory capacity 0.8-1.2vvm, and revolving speed is
300-450r/min, ventilatory capacity, revolving speed and tank pressure by adjusting fermentor are maintained at 20- to control dissolved oxygen level in fermentation liquid
30%.30-48h is converted to get conversion fluid.
(3) product detection
Conversion fluid is collected, is extracted 7 times through ethyl acetate, crystal has been threaded in Rotary Evaporators after merging extract and has gone out
Existing, acetonitrile redissolves crystal and is cleaned by 0.22 μm of organic membrane filter, and filtrate detects 7 α, 15 α-using high performance liquid chromatography
DiOH-DHEA yield.
(4) conversion results
Using A) it is fed in batches technique, the transformation period is reduced to 36h from 52h, and efficiency of pcr product is improved to 53.0%, and cell is dry
Weight peak is improved to 23.5g/L.
3 substrate of embodiment is fed in batches grinding for the hydroxylation reaction that strategy is combined with promotion reducing power regeneration technology to DHEA
Study carefully
(1) C.lini ST-1 cellular liquid culture is prepared
C.lini ST-1 on picking PDA solid medium is inoculated in equipped with 30mL fresh liquid seed culture medium
In 250mL conical flask, 30 DEG C, 120-220r/min culture for 24 hours.Again with the inoculum concentration access of 8-10% (v/v) equipped with fermentation training
In the 5L fermentor for supporting base, liquid amount 3L.In growth phase, cultivation temperature is 25-40 DEG C, and tank pressure is set as 0.05MPa, revolving speed
It is set as 220-300r/min, ventilatory capacity 0.4-1.0vvm, dissolved oxygen level is maintained at 10-20%, grows after 12h to get to suitable
The liquid medium for being suitable for DHEA to convert；
(2) substrate adds technique in batches
Three parts of powdered DHEA of 5g/L accurately are weighed, put into the thallus culture in step (1) respectively in 12h, 16h, 20h
Liquid makes substrate DHEA feed intake final concentration of 15g/L, to reduce the toxic action of high concentration substrate.
(3) promote reducing power regeneration technology
When converting 18h, 15g/L glucose is added as the regenerated nutriment of reducing power, to promote to restore in fermentation liquid
The regeneration of power.
(4) DHEA bioconversion
In the conversion process, cultivation temperature is 25-40 DEG C, and tank pressure is set as 0.05MPa, ventilatory capacity 0.8-1.2vvm, is turned
Speed is 300-450r/min, and dissolved oxygen level in fermentation liquid is controlled by adjusting ventilatory capacity, revolving speed and the tank pressure of fermentor and is kept
In 20-30%.The pH of transformation system is maintained to be by the sodium hydroxide of the acetic acid of fermentor auto-feeding 40% or 10%
6.5,30-48h is converted to get conversion fluid.
(5) product detection
The conversion fluid of step (4) is centrifuged 5-10min, the isometric ethyl acetate of supernatant at 8000-12000r/min
Extracting 7 times precipitates with appropriate chloroform 3 times, has been threaded to crystal appearance in Rotary Evaporators after merging extract, acetonitrile is multiple
Molten crystal is simultaneously cleaned by 0.22 μm of organic membrane filter, and filtrate utilizes 7 α of high-efficient liquid phase chromatogram technique analysis, 15 α-diOH-DHEA
Content.
(6) conversion results
Compared with method for transformation in embodiment 1, the transformation period is reduced to 44h from 52h, and efficiency of pcr product is improved to 66.6%,
Improve 35.6%.Dry cell weight peak is improved from 19.0g/L to 23.9g/L.
Claims (2)
- The regeneration of promotion reducing power generates 7 α to improve C.lini ST-1 hydroxylation DHEA under the conditions of 1. a kind of high concentration DHEA feeds intake, The method of 15 α-diOH-DHEA, its feature is as follows:(1) using on April 24th, 2012 being preserved in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microorganism and grind Study carefully China Committee for Culture Collection of Microorganisms's common micro-organisms center's deposit number be CGMCC No.6501 flax Piercing mould (Colletotrichum lini) ST-1 of disk spore is production strain, and 25-40 DEG C, training is activated under the conditions of 120-220r/min It supports and obtains seed liquor, which is applied in PDA culture medium；(2) the cellular liquid culture of bacterial strain is prepared: the CGMCC No.6501 bacterium on the PDA solid medium of picking step (1) One ring of strain, is inoculated in the sterilized 250mL conical flask equipped with 20-50mL seed culture medium, and cultivation temperature is 25-40 DEG C, It sets on shaking table with the revolving speed culture 18-30h of 120-220r/min to mid log phase to get to CGMCC No.6501 bacterial strain Cellular liquid culture；(3) thalli growth in 5L fermentor: cellular liquid culture the connecing with volume ratio 8-10% that will be prepared in step (2) Kind amount access is equipped in the 5L fermentor of fermentation medium, liquid amount 3L；In growth phase, cultivation temperature is 25-40 DEG C, tank Pressure is set as 0.05MPa, and revolving speed is set as 220-300r/min, ventilatory capacity 0.4-1.0vvm；By adjust fermentor ventilatory capacity, Revolving speed and tank pressure are maintained at 10-20% to control dissolved oxygen level in fermentation liquid, grow after 12h to get converting to being suitable for DHEA Liquid medium；(4) add in batches DHEA and promote reducing power regeneration technology: accurately weigh three parts of powdered DHEA of 5g/L, in 12h, 16h, 20h puts into the bacterial culture fluid in step (3) respectively, and substrate DHEA is made to feed intake final concentration of 15g/L；When converting 18h, disposably Add 15g/L glucose；(5) DHEA bioconversion: in the conversion process, cultivation temperature is 25-40 DEG C, and tank pressure is set as 0.05MPa, and ventilatory capacity is 0.8-1.2vvm, revolving speed 300-450r/min are controlled in fermentation liquid by adjusting ventilatory capacity, revolving speed and the tank pressure of fermentor Dissolved oxygen level is maintained at 20-30%；It maintains to convert by the sodium hydroxide of the acetic acid of fermentor auto-feeding 40% or 10% The pH of system is 6.5, converts 30-48h to get conversion fluid；(6) product detection: the conversion fluid of step (5) is centrifuged 5-10min at 8000-12000r/min, supernatant is in equal volume Ethyl acetate extracts 7 times, precipitates with appropriate chloroform 3 times, has been threaded to crystal in Rotary Evaporators after merging extract and has gone out Existing, acetonitrile redissolves crystal and is cleaned by 0.22 μm of organic membrane filter, and filtrate utilizes 7 α of high-efficient liquid phase chromatogram technique analysis, 15 α- The content of diOH-DHEA.
- 2. the method as described in claim 1, which is characterized in that promote the regenerated conversion work of reducing power using being fed in batches to combine Skill, 7 α of product, 15 α-diOH-DHEA yield improve 35.6% compared with Conventional reformat method, and the transformation period foreshortens to 44h from 52h.
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Citations (2)
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|---|---|---|---|---|
| CN102757997A (en) * | 2012-05-28 | 2012-10-31 | 江南大学 | Method for preparing 7alpha, 15alpha-dihydroxy androstenone by pre-inducing substrate |
| 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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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757997A (en) * | 2012-05-28 | 2012-10-31 | 江南大学 | Method for preparing 7alpha, 15alpha-dihydroxy androstenone by pre-inducing substrate |
| 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 |
Non-Patent Citations (1)
| Title |
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| Improvement of NADPH-dependent P450-mediated biotransformation of 7a,15a-diOH-DHEA from DHEA by a dual cosubstrate-coupled system;Yan Wu、Zheng-Hong Xu等;《Steroids》;20150530;第16页第2节材料与方法，第18页第3.3节，第19页第3.5节，第20页第4节 * |
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Inventor after: Xu Zhenghong Inventor after: Shi Jinsong Inventor after: Li Hui Inventor after: Li Cong Inventor after: Chen Liying Inventor before: Xu Zhenghong Inventor before: Shi Jinsong Inventor before: Li Hui Inventor before: Li Cong |
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| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211206 Address after: 300000 No. 19, Xinye 9th Street, Binhai New Area, Tianjin Patentee after: TIANJIN TIANYAO PHARMACEUTICAL Co.,Ltd. Patentee after: Jiangnan University Address before: No. 1800 road 214122 Jiangsu Lihu Binhu District City of Wuxi Province Patentee before: Jiangnan University |
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| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 300000 No. 19, Xinye 9th Street, Binhai New Area, Tianjin Patentee after: Tianjin Pharmaceutical Co.,Ltd. Patentee after: Jiangnan University Address before: 300000 No. 19, Xinye 9th Street, Binhai New Area, Tianjin Patentee before: TIANJIN TIANYAO PHARMACEUTICAL Co.,Ltd. Patentee before: Jiangnan University |