CN104593444B - A kind of method that fermentation prepares epothilone B - Google Patents
A kind of method that fermentation prepares epothilone B Download PDFInfo
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- CN104593444B CN104593444B CN201310525175.XA CN201310525175A CN104593444B CN 104593444 B CN104593444 B CN 104593444B CN 201310525175 A CN201310525175 A CN 201310525175A CN 104593444 B CN104593444 B CN 104593444B
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Abstract
The invention discloses a kind of methods with active carbon substitution macroreticular resin fermentation synthesis epothilone B, in fermentation process, adsorbent of the activated carbon as target product are added into fermentation medium, and the additive amount of activated carbon is 10-30g in every liter of culture medium.Of the invention is easy to operate, and mild condition, raw material sources are cheap extensively, and equipment requirement is low, reduces production cost, and improve fermentation yield, is particularly suitable for industrialized production.
Description
Technical field
The invention belongs to the production technical fields of epothilone B, and in particular to one kind prepared by fermentation angstrom win it is mould
The method of plain B.
Background technique
Epothilone B (Epothilone B) is that slime bacteria sorangium cellulosum (Sorangium cellulosum) is generated
A kind of polyketone secondary metabolite, be a kind of compound of macrolides, it and clinically widely applied antitumorization
Drug taxol stable micro-pipe activity having the same is treated, and active to the tumour cell of taxol resistance.In addition angstrom rich
Mycin B makes it by it is believed that being a kind of to have more market future than taxanes drug from the feature of microbial metabolic products
Anticancer drug.Epothilone B molecular formula is C27H41NO6S, relative molecular weight 507.68, structural formula is as follows:
Compared with clinical research, the production technology of epothilone B also lags far behind.Its main bottleneck is that fermentation level is low
Under, slime bacteria fermentation at present prepares the yield of epothilone B only in the level of 200mg/L fermentation liquid.This is because epothilone B
And its homologue is larger to the toxicity and feedback inhibition of production bacterial strain, and the fermentation level of epothilone B is caused to be difficult to improve.
In response to the above problems, now commonplace solution is that macroreticular resin such as XAD- is added in the fermentation medium
16, the adsorbed products such as AB-8 or DA201 act on the toxicity inhibition of bacterial strain to reduce product, while stimulating microorganism continuous
Metabolism generates epothilone B.
But this method has the disadvantage in that resin is unevenly distributed in fermentation liquid, and fermentation process sampling is caused to lose ginseng
Examine value；Resin is more empty infiltration substances, has bigger internal surface area, in high-pressure sterilizing course, internal air can not
Sterilizing completely, is easy to cause fermentation failure due to microbiological contamination；The valve of fermentor is easily blocked by resin；Resin price is expensive, increases
Add production cost.
Therefore a kind of material that can substitute resin is found to be of great significance to the industrialization of epothilone B.
Summary of the invention
It is an object of the present invention to generally use macroporous resin adsorption product for current epothilone B zymotechnique
Disadvantage, provides a kind of fermentation level height, and the low method of production cost mainly substitutes macroreticular resin, particular technique with active carbon
Scheme is as follows:
1) seed culture fluid is cultivated in seeding tank；
The seed culture medium preparation method:
Starch 10g/L, milk powder 5g/L, peptone 5g/L, soybean powder 4g/L, magnesium sulfate 1g/L, calcium chloride 1g/L, calcium carbonate
3g/L, pH7.5.
Suitable seed culture medium is prepared, 29-31 DEG C is cooled to after 120 DEG C of sterilizing 30min, by seed culture medium volume ratio
The shake-flask seed in the inoculum concentration access 60-70h kind age of 5-10%, controlled at 29-31 DEG C in incubation, pressure 0.04-
0.06Mpa ventilates and protects in incubation for 0.5-1.0VVM, revolving speed 100-300rpm if the initial dissolved oxygen of seeding tank is 100%
Dissolved oxygen is held 30% or more, persistently cultivates 60-70h.
2) seed culture fluid is accessed in the fermenter, and adds active carbon, and continuing fermentation obtains epothilone B.
The fermentation medium preparation method:
Fermentation medium A: starch 40g/L, milk powder 10g/L, peptone 10g/L, soybean powder 4g/L, magnesium sulfate 1g/L, chlorine
Change calcium 1g/L, calcium carbonate 3g/L, activated carbon 10-30g/L, PH7.5.
The activated carbon is powdered or graininess.
Preferably, activated carbon is 20g/L in fermentation medium A, and activated carbon is powdered.
Suitable fermentation medium A is prepared, 29-31 DEG C is cooled to after 120 DEG C of sterilizing 30min, by fermentation medium volume
Inoculum concentration than 5-10% accesses seed culture fluid, and it is 29-31 DEG C, pressure 0.04-0.06Mpa that temperature is kept in fermentation process,
It ventilates and keeps dissolved oxygen in fermentation process if fermentor initial dissolved oxygen amount is 100% for 0.5-1.0VVM, revolving speed 50-200rpm
30% or more, ferment 11-13 days.
Preferably, seed culture fluid is accessed by the inoculum concentration of fermentation medium volume ratio 8%.
Fermentation medium B: starch 40g/L, milk powder 10g/L, peptone 10g/L, soybean powder 4g/L, magnesium sulfate 1g/L, chlorine
Change calcium 1g/L, calcium carbonate 3g/L, activated carbon 5-10g/L, PH7.5.
The activated carbon is powdered or graininess.
Preferably, activated carbon is 10g/L in fermentation medium B, and activated carbon is powdered.
Suitable fermentation medium B is prepared, 29-31 DEG C is cooled to after 120 DEG C of sterilizing 30min, by fermentation medium volume
Inoculum concentration than 5-10% accesses seed culture fluid, and it is 29-31 DEG C, pressure 0.04-0.06Mpa that temperature is kept in fermentation process,
Ventilation is 0.5-1.0VVM, revolving speed 50-200rpm, if initial dissolved oxygen is 100%, control dissolved oxygen is 30% or more, fermented and cultured
In the process, activated carbon is added in primary or gradation, and adding total amount is 5-20 g/L, is fermented 11-13 days.
Preferably, during fermented and cultured, by sample detection, when fermentation unit reaches 50mg/L or more, addition
The activated carbon of 10g/L；When fermentation unit reaches 100mg/L or more, then add the activated carbon of 10g/L.
Preferably, the activated carbon filled into is powdered.
The sample detection condition are as follows: under 250nm wavelength, mobile phase Yi Jing ︰ water=1 ︰ 1, C18 column, HPLC detection.
It prepares activated carbon feed supplement liquid: activated carbon is poured into feed supplement tank, suitable quantity of water is added, 120 DEG C of sterilizing 30min are cooling
To 29-31 DEG C, for use.
In the present invention, the bacterial strain used that ferments is sorangium cellulosum (Sorangium cellulosum), which can lead to
Cross commercial sources purchase, bacterial strain deposit number: ATCC15384.
Further, above-mentioned fermentation liquid is isolated and purified, obtains the epothilone B of high-purity.
Of the invention is easy to operate, and mild condition, raw material sources are cheap extensively, and equipment requirement is low, reduces life
Cost is produced, and improves fermentation yield.
Especially applicant is found surprisingly that, by the addition activated carbon resin that generally uses in the prior art of substitution as
Adsorbent in fermentation process, not only activated carbon sterilizing is more abundant, reduces the risk of microbiological contamination in fermentation process, and sending out
Under conditions of zymotic fluid is stirred continuously, activated carbon adsorbent is evenly distributed, continuous adsorbed product, reduces product to the toxicity of bacterial strain
Inhibiting effect improves the yield of target product, while the market price of activated carbon is cheaper, extremely suitable industry metaplasia
It produces.
The chromatogram that epothilone B HPLC is detected in 5 fermentation liquid of Detailed description of the invention embodiment, the wherein guarantor of epothilone B
Staying the time is 3.358min.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
And not meaning that has any restrictions to the present invention.By sorangium cellulosum (Sorangium cellulosum) cryo-conservation.Shaking flask
Culture: 2L shaking flask fills 200ml seed culture medium, is cooled to 30 DEG C after 120 DEG C of sterilizing 30min, accesses the strain of cryo-conservation,
30 DEG C, shaking flask culture 60-70h under revolving speed 200rpm.
Embodiment 1 does not add product adsorbent fermenting and producing epothilone B
Fermentation medium I: starch 40g/L, milk powder 10g/L, peptone 10g/L, soybean powder 4g/L, magnesium sulfate 1g/L, chlorine
Change calcium 1g/L, calcium carbonate 3g/L, pH7.5.
3.5L seed culture medium is prepared in 5L seeding tank, 30 DEG C is cooled to after 120 DEG C of sterilizing 30min, by seed culture
The shake-flask seed in the inoculum concentration access 65h kind age of base volume ratio 8%, controlled at 30 DEG C, pressure 0.05Mpa, ventilating is
0.7VVM, revolving speed 100-300rpm control dissolved oxygen 30% or more, persistently cultivate 65h.
35L fermentation medium I is prepared in 50L fermentor, 30 DEG C is cooled to after 120 DEG C of sterilizing 30min, by fermentation medium
The inoculum concentration of volume ratio 8% accesses seed culture fluid, and it is 30 DEG C that temperature is kept in fermentation process, and pressure 0.05Mpa, ventilating is
0.8VVM, revolving speed 50-200rpm keep dissolved oxygen 30% or more in fermentation process, and fermented and cultured 12 days put tank, wavelength
250nm, HPLC detect fermentation unit 5mg/L.
Embodiment 2 adds product adsorbent macroreticular resin fermenting and producing epothilone B
Fermentation medium II: starch 40g/L, milk powder 10g/L, peptone 10g/L, soybean powder 4g/L, magnesium sulfate 1g/L, chlorine
Change calcium 1g/L, calcium carbonate 3g/L, XAD-16 resin 2.0-3.0% (the ratio between resin volume and fermentation medium volume), pH7.5.
3.5L seed culture medium is prepared in 5L seeding tank, 30 DEG C is cooled to after 120 DEG C of sterilizing 30min, by seed culture
The shake-flask seed in the inoculum concentration access 65h kind age of base volume ratio 8%, controlled at 30 DEG C, pressure 0.05Mpa, ventilating is
0.7VVM, control revolving speed control dissolved oxygen 30% or more, persistently cultivate 65h in 100-300rpm.
35L fermentation medium II is prepared in 50L fermentor, is cooled to 30 DEG C after 120 DEG C of sterilizing 30min, by fermentation training
The inoculum concentration for supporting II volume ratio 8% of base accesses seed culture fluid, and it is 30 DEG C, pressure 0.05Mpa that temperature is kept in fermentation process,
Ventilating is 0.8VVM, revolving speed 50-200rpm, keeps dissolved oxygen 30% or more in fermentation process, fermented and cultured 12 days put tank, wave
Long 250nm, HPLC detect fermentation unit 110mg/L.
Embodiment 3 adds product adsorbent powdery activated carbon fermenting and producing epothilone B
3.5L seed culture medium is prepared in 5L seeding tank, 29 DEG C is cooled to after 120 DEG C of sterilizing 30min, by seed culture
The shake-flask seed in the inoculum concentration access 70h kind age of base volume ratio 5%, controlled at 31 DEG C, pressure 0.04Mpa, ventilating is
0.5VVM, revolving speed 100-300rpm, control dissolved oxygen continue incubation time 60h 30% or more.
35L fermentation medium A is prepared in 50L fermentor, wherein activated carbon is powdered activated carbon, 120 DEG C of sterilizing 30min
After be cooled to 29 DEG C, access seed culture fluid by the inoculum concentration of fermentation medium volume ratio 5%, keep in fermentation process the temperature to be
It 31 DEG C, pressure 0.04Mpa, ventilating as 0.5VVM, revolving speed 50-200rpm keeps in fermentation process dissolved oxygen 30% or more,
Tank is put within fermented and cultured 13 days, wavelength 250nm, HPLC detect fermentation unit 130mg/L.
Embodiment 4 adds product absorbent particles shape activated carbon fermenting and producing epothilone B
3.5L seed culture medium is prepared in 5L seeding tank, 31 DEG C is cooled to after 120 DEG C of sterilizing 30min, by seed culture
The shake-flask seed in the inoculum concentration access 60h kind age of base volume ratio 10%, controlled at 29 DEG C, pressure 0.06Mpa, ventilating is
1.0VVM, revolving speed 100-300rpm control dissolved oxygen 30% or more, persistently cultivate 70h.
35L fermentation medium A is prepared in 50L fermentor, wherein activated carbon is granule activated carbon, 120 DEG C of sterilizings
It is cooled to 31 DEG C after 30min, accesses seed culture fluid by the inoculum concentration of fermentation medium volume ratio 10%, is kept in fermentation process
Temperature is 29 DEG C, pressure 0.06Mpa, is ventilated as 1.0VVM, revolving speed 50-200rpm, keeps dissolved oxygen 30% in fermentation process
More than, tank is put within fermented and cultured 11 days, wavelength 250nm, HPLC detect fermentation unit 105mg/L.
Product adsorbent powdery activated carbon fermenting and producing epothilone B is added batch-wise in embodiment 5
3.5L seed culture medium is prepared in 5L seeding tank, 29 DEG C is cooled to after 120 DEG C of sterilizing 30min, by seed culture
The shake-flask seed in the inoculum concentration access 60h kind age of base volume ratio 5%, controlled at 29 DEG C, pressure 0.04Mpa, ventilating is
0.5VVM, revolving speed 100-300rpm control dissolved oxygen 30% or more, persistently cultivate 60h.
35L fermentation medium B is prepared in 50L fermentor, wherein activated carbon is powdered activated carbon, 120 DEG C of sterilizing 30min
After be cooled to 29 DEG C, access seed culture fluid by the inoculum concentration of fermentation medium volume ratio 10%, keep in fermentation process the temperature to be
29 DEG C, pressure 0.04Mpa, ventilate as 0.5VVM, control revolving speed kept in 50-200rpm, fermentation process dissolved oxygen 30% with
On, sample detection when fermentation unit reaches 50mg/L, adds the activated carbon of 10g/L, when fermentation unit reaches 100mg/L, then
The activated carbon for adding 10g/L, continues to ferment, and tank is put in fermentation on the 11st day, and wavelength 250nm, HPLC detect fermentation unit 145mg/L.
Product absorbent particles shape activated carbon fermenting and producing epothilone B is added batch-wise in embodiment 6
3.5L seed culture medium is prepared in 5L seeding tank, 31 DEG C is cooled to after 120 DEG C of sterilizing 30min, by seed culture
The shake-flask seed in the inoculum concentration access 70h kind age of base volume ratio 10%, controlled at 31 DEG C, pressure 0.06Mpa, ventilating is
1.0VVM, revolving speed 100-300rpm control dissolved oxygen 30% or more, persistently cultivate 70h.
35L fermentation medium B is prepared in 50L fermentor, wherein activated carbon is granule activated carbon, 120 DEG C of sterilizings
It is cooled to 31 DEG C after 30min, accesses seed culture fluid by the inoculum concentration of fermentation medium volume ratio 5%, keeps temperature in fermentation process
Degree is 31 DEG C, pressure 0.06Mpa, is ventilated as 1.0VVM, revolving speed 50-200rpm, kept in fermentation process dissolved oxygen 30% with
On, when fermentation unit reaches 50mg/L, the activated carbon of 10g/L is added, when fermentation unit reaches 100mg/L, then is added
The activated carbon of 10g/L continues to ferment, and tank is put in fermentation on the 13rd day, and wavelength 250nm, HPLC detect fermentation unit 120mg/L.
Product absorbent particles shape activated carbon fermenting and producing epothilone B is added batch-wise in 5L seeding tank in embodiment 7
3.5L seed culture medium is prepared, 31 DEG C is cooled to after 120 DEG C of sterilizing 30min, is connect by the inoculum concentration of seed culture medium volume ratio 10%
Enter the shake-flask seed in 70h kind age, controlled at 31 DEG C, pressure 0.06Mpa, ventilates as 1.0VVM, revolving speed 100-
300rpm controls dissolved oxygen 30% or more, persistently cultivates 70h.
35L fermentation medium B is prepared in 50L fermentor, wherein activated carbon is granule activated carbon, 120 DEG C of sterilizings
It is cooled to 31 DEG C after 30min, accesses seed culture fluid by the inoculum concentration of fermentation medium volume ratio 5%, keeps temperature in fermentation process
Degree is 31 DEG C, pressure 0.06Mpa, is ventilated as 1.0VVM, revolving speed 50-200rpm, kept in fermentation process dissolved oxygen 30% with
On, when fermentation unit reaches 80mg/L, the activated carbon of 20g/L is added, continues to ferment, fermentation puts tank, wavelength on the 13rd day
250nm, HPLC detect fermentation unit 125mg/L.
It should be noted that the foregoing is merely illustrative of the preferred embodiments of the present invention, it is not intended to restrict the invention, it is all
Made any modifications, equivalent replacements, and improvements etc. within the spirit and principles in the present invention should be included in guarantor of the invention
Within the scope of shield.
Claims (4)
1. a kind of method for preparing epothilone B with fermentation, which is characterized in that the fermentation process includes:
1) seed culture fluid is cultivated in seeding tank；
2) seed culture fluid is accessed in the fermenter, and adds active carbon, and continuing fermentation obtains epothilone B；
The active carbon is powdered or graininess, wherein when active carbon is powdered, prepared by the active carbon
It is added when fermentation medium, additive amount is the active carbon that every liter of culture medium adds 10-30g；Alternatively, when preparing fermentation medium
Addition, additive amount are the active carbon that every liter of culture medium adds 5-10g, add active carbon again or by several times in fermentation process, mend
Aggregation amount is the active carbon that every liter of culture medium adds 5-20g；
Wherein, when active carbon is graininess, the active carbon, the addition when preparing fermentation medium, additive amount is every liter
Culture medium adds the active carbon of 5-10g, adds active carbon again or by several times in fermentation process, adding total amount is every liter of culture medium
Add the active carbon of 5-20g；
Wherein, fermentation bacterial strain uses therefor is slime bacteria sorangium cellulosum.
2. the method as described in claim 1, which is characterized in that the active carbon be it is powdered, preparing fermentation medium
Shi Tianjia, additive amount are the activated carbon that every liter of culture medium adds 5-10g；Activated carbon is added again or by several times in fermentation process,
Adding total amount is the activated carbon that every liter of culture medium adds 5-20g.
3. as described in claim 1, which is characterized in that step 1) accesses strain into seeding tank, and condition of culture temperature is 29-
It 31 DEG C, pressure 0.04-0.06Mpa, ventilates as 0.5-1.0VVM, revolving speed 100-300rpm, if seeding tank initial dissolved oxygen amount
It is 100%, keeps dissolved oxygen 30% or more in incubation, incubation time 60-70h.
4. as described in claim 1, which is characterized in that fermentation tank culture condition is by fermentation medium volume in step 2)
5-10% inoculum concentration accesses seed culture fluid, and fermentation temperature is 29-31 DEG C, and pressure 0.04-0.06Mpa ventilates as 0.5-
1.0VVM, revolving speed 50-200rpm, if fermentor initial dissolved oxygen amount is 100%, keep in incubation dissolved oxygen 30% with
On, it ferments 11-13 days.
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| CN102633792A (en) * | 2011-02-15 | 2012-08-15 | 天津尚德药缘科技有限公司 | Method for preparing epothilone D and B |
| CN102373252B (en) * | 2011-11-04 | 2013-11-27 | 陕西科技大学 | Fermentation production process of Epothilone B |
| CN102586358B (en) * | 2012-01-11 | 2013-06-12 | 湖北宏中药业有限公司 | Biosynthesis method for improving yield of epothilone B |
| CN103146594B (en) * | 2012-11-08 | 2015-04-01 | 山东轻工业学院 | Sorangiumcellulosum strain and application thereof to synthesis of epothilone |
| CN103243134B (en) * | 2013-04-15 | 2015-04-22 | 陕西科技大学 | Fermentation production method based on epothilone B metabolic pathways |
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