CN103937851A - Method for producing epothilone B based on coupling of microbial fermentation and membrane separation techniques - Google Patents

Method for producing epothilone B based on coupling of microbial fermentation and membrane separation techniques Download PDF

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CN103937851A
CN103937851A CN201410165832.9A CN201410165832A CN103937851A CN 103937851 A CN103937851 A CN 103937851A CN 201410165832 A CN201410165832 A CN 201410165832A CN 103937851 A CN103937851 A CN 103937851A
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membrane
epothilone
fermentation
filtration
nanofiltration
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龚国利
马利云
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Shaanxi University of Science and Technology
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Shaanxi University of Science and Technology
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Abstract

The invention discloses a method for producing epothilone B based on coupling of microbial fermentation and membrane separation techniques. The invention proposes a separation and fermentation coupling method mainly aiming at the problems that toxicity and feedback inhibition on cells are generated due to increase of a product epothilone B in the fermentation process, and bacteria growth in the fermentation process is affected. The method comprises the following steps: fermenting and cultivating, filtering by a micro-filtration membrane, carrying out ultrafiltration and nanofiltration, feeding materials, concentrating and extracting. The concentration of the fermentation liquor product epothilone B is reduced to a lower level, the growth inhibition action on the strain is relieved, the yield of the epothilone B is improved, the production cost is reduced, post-treatment of a fermentation liquor and refining of the product are facilitated, and large-scale production becomes possible, so that the method has a good industrial production prospect.

Description

A kind of method based on microorganism fermentation and membrane separation technique coupling production epothilone B
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of method based on microorganism fermentation and membrane separation technique coupling production epothilone B.
Background technology
Ebormycine (epothilones) is the class secondary metabolite that slime bacteria sorangium cellulosum (Sorangium cellulosum) produces, by the G.H & ouml of the national biotechnology center of Germany (GBF), the people such as fle were in reported first in 1993.Its relative molecular mass is 585.There is the stable effect of tubulin polymerization and microtubule, taxol and other cancer therapy drug are had the cancer cells of resistance and have very high activity; Have stronger resistance and be convenient to preparation and use, therefore ebormycine, as the PTS that has potentiality, has caused the research enthusiasm of global biologist and pharmacy man.An at present existing ebormycine medicine (BMS-247550, Ixabepilone, for the treatment of ovarian cancer) put on market, at least also have in addition six ebormycine medicine (ABJ-879, Patupilone, BMS-310705, KOS-862, KOS-1584 and ZK-EPO) carrying out clinical study.Ebormycine is expected to become the more efficiently antitumor drug that replaces taxol.
The main microorganism fermentative production that relies on obtains ebormycine at present, before to separation of fermentative broth ebormycine, need to remove the thalline in sorangium cellulosum fermented liquid, a step is not only in thalline separation affects the key operation of product separation effect, and due to the degrading enzyme that contains ebormycine in the thalline in fermented liquid, as separated not in time, will produce degraded to the ebormycine in fermented liquid, affect product yield.
Micro-filtration (MF) is widely used in cell, bacterium separates with atomic, and the scope of object ion is 0.01 μ m～10 μ m.Taking static pressure difference as impellent, utilize the sieving action of film to separate, its effect is equivalent to filter.Because porosity accounts for 70%～80% of cumulative volume, therefore resistance is very little, filtration velocity is very fast.
Ultrafiltration (UF) is a kind of screening and separating process, under the impellent effect of static pressure difference, solvent in stock liquid and little solute particles see through film to low-tension side from the feed liquid side of high pressure, be commonly referred to as light liquid or see through liquid, and macroparticle component tunicle stops, they are increased the concentration in the surplus liquid of filter.Ultrafiltration is mainly used in separating macromolecular cpd (protein, nucleic acid polymers, starch, natural gum, enzyme etc.) from liquid phase substance, thereby reaches concentrating, separate, purify, purifying of some solution that contains polymer substance (protein, enzyme, virus).
The same with the sepn process such as ultrafiltration and reverse osmosis, nanofiltration is also the sepn process taking pressure difference as impellent, is a non-reversible process.Its separation mechanism can use charge model (space charge model and fixed charge model), pore model and just Coulomb repulsion and the steric hindrance model etc. of proposition are described in recent years.Compared with other membrane sepn process, an advantage of nanofiltration membrane is the organism that can hold back through the small molecules amount of ultra-filtration membrane, and the part inorganic salt that the reverse osmosis membrane of dialysing is again held back, namely can make " concentrating " to synchronize and carry out with desalination.
The production of ebormycine and exploitation are limited to laboratory level and pilot scale level at present.The laboratory on a small scale key step of preparation comprises: microorganism fermentation, absorption with macroporous adsorbent resin, resolves and separates, the dry crude product that obtains, by this method, ebormycine constantly accumulates, and fermented liquid concentration constantly increases, the sharply decline of dissolved oxygen, thalline death, causing the activity of ebormycine synthetic enzyme to decline, there is stagnating even downtrending in the accumulation of the ebormycine in fermented liquid, and output is difficult to further improve.And the operation of unit is all relatively independent in Laboratory Production preparation process, the production time extends, and production efficiency reduces, and cost increases.Also application of membrane separation technology is not arrived in the middle of the production of ebormycine at present.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of method based on microorganism fermentation and membrane separation technique coupling production epothilone B is provided, its output is high, and simple to operate, has shortened the production time, has reduced cost.
For achieving the above object, the technical solution used in the present invention is as follows:
1) fermentation culture: in 1L inoculation 10～100mg bacterial classification ratio, sorangium cellulosum is inoculated in fermentor tank and is fermented;
2) micro-filtrate membrane filtration: in fermenting process in the time that the output of epothilone B reaches 0.1mg/L, the fermented liquid in fermentor tank is filtered by the micro-filtration with microfiltration membrane, the fermented liquid of being held back by microfiltration membrane turns back in fermentor tank, continues fermentation, and filtered solution enters concentration basin;
3) ultra-filtration membrane and nanofiltration membrane are filtered: when the liquid bulk in concentration basin accumulate fermentor tank volume 50%～60% time, liquid in concentration basin is filtered by the ultrafiltration instrument with ultra-filtration membrane with the nanofiltration instrument of nanofiltration membrane, turn back in fermentor tank through the trapped fluid of ultra-filtration membrane, filtered solution continues to filter through nanofiltration membrane; Trapped fluid after nanofiltration membrane returns to concentration basin, and filtered solution turns back in fermentor tank;
4) the concentrated of epothilone B slightly carried: the liquid in concentration basin is concentrated, extracted, obtain epothilone B.
Described sorangium cellulosum is ATCC25532 or ATCC25569.
Described step 1) in the pH value of fermention medium that adopts of fermentation be 7.2～7.5, and at 115 DEG C sterilizing 30min; Fermentation culture based component is yeast powder 20～50g/L, W-Gum 5～10g/L, SODIUM PHOSPHATE, MONOBASIC 1～2g/L, Sodium phosphate dibasic 1～2g/L, MgSO 47H 2o2～5g/L, FeSO 47H 2o0.1～0.5g/L, CaCl 22～5g/L, MnCl 20.1～0.5g/L and glucose 10～15g/L; Fermentation condition is: temperature is 28～32 DEG C, and mixing speed is 100～150r/min, and air flow is 0.3～0.6vvm.
During the fermentation the fresh culture identical with fermentation culture based component is pumped into and in fermentor tank, carries out feed supplement; Liquid amount in fermentor tank is 60～75%.
Hyperfiltration membrane assembly in micro-filtration membrane module, ultrafiltration instrument in described micro-filtration is plate-type flat facial mask, tubular type, rolling or tubular fibre formula, and the nanofiltration membrane component in nanofiltration instrument is rolling.
Described microfiltration membrane is cellulose acetate film, poly (ether sulfone) film or ceramic membrane, and aperture is 0.8 μ m, 1.0 μ m or 1.2 μ m; Ultra-filtration membrane is polyvinylidene fluoride film, hollow-fibre membrane or polyacrylonitrile film, and the molecular weight that dams is 1-150KD; Nanofiltration membrane is hollow fiber composite membrane, and the molecular weight that dams is 200～2000.
Described step 2) in by the condition of filtering with the micro-filtration of microfiltration membrane be: temperature is 30～40 DEG C, and pressure is 0.2～0.5MPa.
Described step 3) in ultrafiltration instrument by with the ultra-filtration membrane operational condition of filtering be: temperature is 30～40 DEG C, and pressure is 0.2～0.5MPa.
Described step 3) in nanofiltration instrument by with the nanofiltration membrane condition of filtering be: temperature is 30～40 DEG C, and pressure is 0.2～0.5MPa.
Described step 4) in extraction be to adopt the mixture of methyl alcohol and acetic acid to carry out, and in the mixture of methyl alcohol and acetic acid, the volume ratio of methyl alcohol and acetic acid is 7:3～9:1.
Compared with prior art, the beneficial effect that the present invention has: when sorangium cellulosum fermentation produces epothilone B, utilize membrane separation technique that the ebormycine in fermented liquid is separated concentrated in time, make production concentration in fermented liquid maintain all the time lower level, reduce the restraining effect of product epothilone B to thalline, make the activity of sorangium cellulosum epothilone B synthetase series maintain all the time higher level, further improve ebormycine fermentation yield.And, the present invention is based on microorganism fermentation and membrane separation technique and be coupled, realize the operate continuously that microbe transformation method is produced ebormycine, be conducive to the aftertreatment of fermentation and refining of target product, improve production efficiency, reduce production cost; Behaviour's mild condition of the present invention, good stability, rapidly and efficiently, equipment and simple to operate, has shortened the production time, and energy consumption is low, can be applied to the suitability for industrialized production of epothilone B.
Brief description of the drawings
Fig. 1 is schematic flow sheet of the present invention;
In figure, 1 is feed supplement bottle, and 2 is the first pump, and 3 is fermentor tank, and 4 is the second pump, and 5 is filtration unit, and 6 is concentration basin, and 7 is the 3rd pump, and 8 is ultrafiltration instrument, and 9 is nanofiltration instrument.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in detail.
Referring to Fig. 1, the present invention relates to a kind of device based on microorganism fermentation and membrane separation technique coupling production epothilone B, comprise feed supplement bottle 1, the first pump 2, fermentor tank 3, the second pump 4, filtration unit 5 with microfiltration membrane, concentration basin 6, the 3rd pump 7, with the ultrafiltration instrument 8 of ultra-filtration membrane with the nanofiltration instrument 9 of nanofiltration membrane; Wherein, the entrance of fermentor tank 3 connects feed supplement bottle 1, and the first pump 2 is set between feed supplement bottle 1 and fermentor tank 3; The outlet of fermentor tank 3 is connected with the entrance of filtration unit 5 through the second pump 4, the outlet of filtration unit 5 is divided into two-way, one tunnel is connected with the entrance of fermentor tank 3 through pipeline, another road is connected with concentration basin 6 through pipeline, concentration basin 6 is connected with the entrance of ultrafiltration instrument 8 through the 3rd pump 7, the outlet of ultrafiltration instrument 8 is connected with the entrance of fermentation tube 3, the entrance of nanofiltration instrument 9 respectively, and the outlet of nanofiltration instrument 9 is connected with entrance, the concentration basin 6 of fermentor tank 3 respectively.
Embodiment 1
Referring to Fig. 1, the present invention includes following steps:
1) fermentation culture: in 1L inoculation 100mg bacterial classification ratio, sorangium cellulosum ATCC25532 is inoculated in 5L fermentor tank, liquid amount is 60%.Fermentation culture based component is yeast powder 20g/L, W-Gum 5g/L, SODIUM PHOSPHATE, MONOBASIC 1g/L, Sodium phosphate dibasic 1g/L, MgSO 47H 2o2g/L, FeSO 47H 2o0.1g/L, CaCl 22g/L, MnCl 20.1g/L and glucose 10g/L.Fermention medium pH value is 7.2, and at 115 DEG C sterilizing 30min.In fermentor tank 3, fermentation condition is: temperature is 30 DEG C, and mixing speed is 100r/min, and air flow is 0.3vvm.
2) micro-filtrate membrane filtration: after fermentation starts, epothilone B constantly accumulates, fermented liquid concentration constantly increases, in the time that the output of epothilone B reaches 0.1mg/L, start the second pump 4, with the flow velocity of 10mL/min, part fermentation culture is pumped in the filtration unit 5 with microfiltration membrane and filtered, trapped fluid turns back in fermentor tank 3, filtered solution enters in concentration basin 6, and degerming rate reaches 94%.Wherein, microfiltration membrane is cellulose acetate film, and aperture is 1.2 μ m, and micro-filtration membrane module is plate-type flat facial mask, and filtration temperature is 37 DEG C, and pressure is 0.2MPa.
3) ultra-filtration membrane and nanofiltration membrane are filtered: when the filtered solution volume after the degerming in concentration basin 6 reach fermentor tank 3 volumes 50% time, open the 3rd pump 7, flow velocity with 10mL/min pumps into filtered solution in the ultrafiltration instrument 8 with ultra-filtration membrane, its trapped fluid turns back in fermentor tank 3, filtered solution continues through filtering with the nanofiltration instrument 9 of nanofiltration membrane, trapped fluid after filtration returns to concentration basin 6, does not return in fermentor tank 3 containing macromolecular filtered solution.Wherein ultra-filtration membrane is polyvinylidene fluoride film, and molecular weight cut-off is 100KD, and hyperfiltration membrane assembly is rolling, and filtration temperature is 37 DEG C, and pressure is 0.3MPa; Nanofiltration membrane is hollow fiber composite membrane, and molecular weight cut-off is 600, and nanofiltration membrane component is rolling, and filtration temperature is 37 DEG C, pressure 0.5MPa.
4) feed supplement bottle 1 is equipped with the fresh culture of sterilizing, pumps in fermentor tank 3, for the metabolism and growth of cell provides necessary nutrition by the first pump 2 with the speed of 1.0mL/L.Fresh culture pH value is 7.2, and at 115 DEG C sterilizing 30min; Fresh culture based component is: yeast powder 20g/L, W-Gum 5g/L, SODIUM PHOSPHATE, MONOBASIC 1g/L, Sodium phosphate dibasic 1g/L, MgSO 47H 2o2g/L, FeSO 47H 2o0.1g/L, CaCl 22g/L, MnCl 20.1g/L and glucose 10g/L.
5) trapped fluid in concentration basin 6 is concentrated, concentrated volume reach fermentor tank volume 10% time, then the mixture that adds methyl alcohol and acetic acid extracts (methyl alcohol and acetic acid volume ratio are 9:1); Soln using rotary evaporating device evaporation after extraction, obtains epothilone B after evaporate to dryness solution.
This method is produced epothilone B, and after fermentation ends, the accumulation volume of epothilone B reaches 42mg/L, and compared with not adopting batch fermentation of separation coupling, output has improved 40%.
Embodiment 2
Referring to Fig. 1, the present invention includes following steps:
1) with embodiment 1.
2) micro-filtrate membrane filtration: after fermentation starts, epothilone B constantly accumulates, fermented liquid concentration constantly increases, in the time that the output of epothilone B reaches 0.1mg/L, start the second pump 4, with the flow velocity of 10mL/min, part fermentation culture is pumped in the filtration unit 5 with microfiltration membrane and filtered, the fermented liquid being trapped turns back in fermentor tank 3, filtered solution enters in concentration basin 6, and degerming rate reaches 96%; Wherein, microfiltration membrane is poly (ether sulfone) film, and aperture is 1.0 μ m, and micro-filtration membrane module is plate-type flat facial mask, and filtration temperature is 37 DEG C, and pressure is 0.5MPa.
3) with embodiment 1;
4) with embodiment 1;
5) with embodiment 1;
This method is produced epothilone B, and after fermentation ends, the accumulation volume of epothilone B reaches 47mg/L, and compared with not adopting batch fermentation of separation coupling, output has improved 57%.
Embodiment 3
1) with embodiment 1;
2) with embodiment 1;
3) ultra-filtration membrane and nanofiltration membrane filtering and concentrating: when the filtered solution volume after the degerming in concentration basin reach fermentor tank volume 55% time, open the 3rd pump 7, flow velocity with 10mL/min pumps into filtered solution in the ultrafiltration instrument 8 with ultra-filtration membrane, its trapped fluid turns back in fermentor tank 3, filtered solution continues through filtering with the nanofiltration instrument 9 of nanofiltration membrane, trapped fluid after filtration returns to concentration basin 6, does not return in fermentor tank 3 containing macromolecular filtered solution; Wherein, ultra-filtration membrane is hollow-fibre membrane, and molecular weight cut-off is 150KD, and hyperfiltration membrane assembly is tubular type, and filtration temperature is 37 DEG C, and pressure is 0.5MPa.Nanofiltration membrane is hollow fiber composite membrane, and molecular weight cut-off is 600, and nanofiltration membrane component is rolling, and filtration temperature is 37 DEG C, and pressure is 0.5MPa.
4) with embodiment 1.
5) with embodiment 1.
This method is produced epothilone B, and after fermentation ends, the accumulation volume of epothilone B reaches 56mg/L, and compared with not adopting batch fermentation of separation coupling, output has improved 87%.
Embodiment 4
1) with embodiment 1;
2) with embodiment 1;
3) ultra-filtration membrane and nanofiltration membrane filtering and concentrating: in the time that the filtered solution volume after the degerming in concentration basin reaches 2.5L, open the 3rd pump 7, flow velocity with 10mL/min pumps into filtered solution in the ultrafiltration instrument 8 with ultra-filtration membrane, its trapped fluid turns back in fermentor tank 3, filtered solution continues through filtering with the nanofiltration instrument 9 of nanofiltration membrane, trapped fluid after filtration returns to concentration basin 6, does not return in fermentor tank 3 containing macromolecular filtered solution; Wherein, ultra-filtration membrane is polyvinylidene fluoride film, and molecular weight cut-off is 100KD, and hyperfiltration membrane assembly is rolling, and the temperature of filtration is 37 DEG C, and pressure is 0.3MPa; Nanofiltration membrane is hollow fiber composite membrane, and molecular weight cut-off is 1000, and nanofiltration membrane component is rolling, and filtration temperature is 37 DEG C, and pressure is 0.7MPa.
4) with embodiment 1.
5) with embodiment 1.
This method is produced epothilone B, and after fermentation ends, the accumulation volume of epothilone B reaches 52mg/L, and compared with not adopting batch fermentation of separation coupling, output has improved 73%.
Embodiment 5
1) fermentation culture: in 1L inoculation 10mg bacterial classification ratio, sorangium cellulosum ATCC25569 is inoculated in 5L fermentor tank, liquid amount is 75%.Fermentation culture based component is yeast powder 50g/L, W-Gum 8g/L, SODIUM PHOSPHATE, MONOBASIC 2g/L, Sodium phosphate dibasic 1.5g/L, MgSO 47H 2o3g/L, FeSO 47H 2o0.3g/L, CaCl 25g/L, MnCl 20.4g/L and glucose 15g/L, fermention medium pH value is 7.2, and at 115 DEG C sterilizing 30min.In fermentor tank 3, fermentation condition is 28 DEG C of temperature, mixing speed 100r/min, air flow 0.3vvm.
2) micro-filtrate membrane filtration: after fermentation starts, epothilone B constantly accumulates, fermented liquid concentration constantly increases, in the time that the output of epothilone B reaches 0.1mg/L, start the second pump 4, with the flow velocity of 10mL/min, part fermentation culture is pumped in the filtration unit 5 with microfiltration membrane and filtered, trapped fluid turns back in fermentor tank 3, filtered solution enters in concentration basin 6, and degerming rate reaches 96%; Wherein, microfiltration membrane is ceramic membrane, and aperture is 0.8 μ m, and micro-filtration membrane module is plate-type flat facial mask, and filtration temperature is 30 DEG C, and pressure is 0.3MPa.The fermented liquid being trapped turns back in fermentor tank 3, and filtered solution enters concentration basin 6, and degerming rate reaches 97%.
3) ultra-filtration membrane and nanofiltration membrane are filtered: when the filtered solution volume after the degerming in concentration basin 6 reach fermentor tank volume 50% time, open the 3rd pump 7, the 3rd pump 7 flow velocitys are 10mL/min, and filtered solution is pumped into ultrafiltration instrument 8, and ultra-filtration membrane is polyacrylonitrile film, molecular weight cut-off is 1KD, hyperfiltration membrane assembly is rolling, and filtration temperature is 40 DEG C, and pressure is 0.2MPa, its trapped fluid turns back in fermentor tank 3, and filtered solution continues to filter, concentrate through nanofiltration instrument 9.Wherein, nanofiltration membrane is hollow fiber composite membrane, and molecular weight cut-off is 200, and nanofiltration membrane component is rolling, and filtration temperature is 35 DEG C, pressure 0.2MPa.Trapped fluid after nanofiltration returns to concentration basin 6, does not return in fermentor tank 3 containing macromolecular filtered solution.
4) feed supplement bottle 1 is equipped with the fresh culture of sterilizing, pumps in fermentor tank, for the metabolism and growth of cell provides necessary nutrition by the first pump 2 with the speed of 1.0mL/L.Fresh culture pH value is 7.2, and at 115 DEG C sterilizing 30min; Fresh culture based component is: yeast powder 50g/L, W-Gum 8g/L, SODIUM PHOSPHATE, MONOBASIC 2g/L, Sodium phosphate dibasic 1.5g/L, MgSO 47H 2o3g/L, FeSO 47H 2o0.3g/L, CaCl 25g/L, MnCl 20.4g/L and glucose 15g/L, fermention medium pH value is 7.2, and at 115 DEG C sterilizing 30min.
5) trapped fluid in concentration basin 6 is concentrated, concentrated volume reach fermentor tank volume 10% time, then the mixture that adds methyl alcohol and acetic acid extracts (methyl alcohol and acetic acid volume ratio are 7:3); Soln using rotary evaporating device evaporation after extraction, obtains epothilone B after evaporate to dryness solution.
This method is produced epothilone B, and after fermentation ends, the accumulation volume of epothilone B reaches 40mg/L, and compared with not adopting batch fermentation of separation coupling, output has improved 33%.
Embodiment 6
1) fermentation culture: in 1L inoculation 40mg bacterial classification ratio, sorangium cellulosum ATCC25532 is inoculated in 5L fermentor tank, liquid amount is 65%.Fermentation culture based component is yeast powder 35g/L, W-Gum 10g/L, SODIUM PHOSPHATE, MONOBASIC 1.5g/L, Sodium phosphate dibasic 2g/L, MgSO 47H 2o5g/L, FeSO 47H 2o0.5g/L, CaCl 23g/L, MnCl 20.5g/L and glucose 13g/L, fermention medium pH value is 7.4, and at 115 DEG C sterilizing 30min.In fermentor tank 3, fermentation condition is 32 DEG C of temperature, mixing speed 150r/min, air flow 0.4vvm.
2) micro-filtrate membrane filtration: after fermentation starts, epothilone B constantly accumulates, fermented liquid concentration constantly increases, in the time that the output of epothilone B reaches 0.1mg/L, start the second pump 4, with the flow velocity of 10mL/min, part fermentation culture is pumped in the filtration unit 5 with microfiltration membrane and filtered, trapped fluid turns back in fermentor tank 3, filtered solution enters in concentration basin 6, and degerming rate reaches 96%; Wherein, microfiltration membrane is cellulose acetate film, and aperture is 1.2 μ m, and micro-filtration membrane module is plate-type flat facial mask, and filtration temperature is 40 DEG C, and pressure is 0.4MPa.The fermented liquid being trapped turns back in fermentor tank 3, and filtered solution enters concentration basin 6, and degerming rate reaches 95%.
3) ultra-filtration membrane and nanofiltration membrane are filtered: when the filtered solution volume after the degerming in concentration basin 6 reach fermentor tank volume 60% time, open the 3rd pump 7, the 3rd pump 7 flow velocitys are 10mL/min, filtered solution is pumped into ultrafiltration instrument 8, and ultra-filtration membrane is polyvinylidene fluoride film, and molecular weight cut-off is 20KD, hyperfiltration membrane assembly is rolling, filtration temperature is 33 DEG C, and pressure is that its trapped fluid of 0.4MPa turns back in fermentor tank 3, and filtered solution continues to filter, concentrate through nanofiltration instrument 9.Wherein, nanofiltration membrane is hollow fiber composite membrane, and molecular weight cut-off is 2000, and nanofiltration membrane component is rolling, and filtration temperature is 30 DEG C, pressure 0.3MPa.Trapped fluid after nanofiltration returns to concentration basin 6, does not return in fermentor tank 3 containing macromolecular filtered solution.
4) feed supplement bottle 1 is equipped with the fresh culture of sterilizing, pumps in fermentor tank, for the metabolism and growth of cell provides necessary nutrition by the first pump 2 with the speed of 1.0mL/L.Fresh culture pH value is 7.4, and at 115 DEG C sterilizing 30min; Fresh culture based component is: yeast powder 35g/L, W-Gum 10g/L, SODIUM PHOSPHATE, MONOBASIC 1.5g/L, Sodium phosphate dibasic 2g/L, MgSO 47H 2o5g/L, FeSO 47H 2o0.5g/L, CaCl 23g/L, MnCl 20.5g/L and glucose 13g/L, fermention medium pH value is 7.2, and at 115 DEG C sterilizing 30min.5) trapped fluid in concentration basin 6 is concentrated, concentrated volume reach fermentor tank volume 10% time, then the mixture that adds methyl alcohol and acetic acid extracts (methyl alcohol and acetic acid volume ratio are 5:1); Soln using rotary evaporating device evaporation after extraction, obtains epothilone B after evaporate to dryness solution.
This method is produced epothilone B, and after fermentation ends, the accumulation volume of epothilone B reaches 45mg/L, and compared with not adopting batch fermentation of separation coupling, output has improved 50%.
Embodiment 7
1) fermentation culture: in 1L inoculation 70mg bacterial classification ratio, sorangium cellulosum ATCC25569 is inoculated in 5L fermentor tank, liquid amount is 70%.Fermentation culture based component is yeast powder 50g/L, W-Gum 8g/L, SODIUM PHOSPHATE, MONOBASIC 2g/L, Sodium phosphate dibasic 1.5g/L, MgSO 47H 2o3g/L, FeSO 47H 2o0.3g/L, CaCl 25g/L, MnCl 20.4g/L and glucose 15g/L, fermention medium pH value is 7.5, and at 115 DEG C sterilizing 30min.Fermentor tank 3 fermentation conditions are 30 DEG C of temperature, mixing speed 120r/min, air flow 0.6vvm.
2) micro-filtrate membrane filtration: after fermentation starts, epothilone B constantly accumulates, fermented liquid concentration constantly increases, in the time that the output of epothilone B reaches 0.1mg/L, start the second pump 4, with the flow velocity of 10mL/min, part fermentation culture is pumped in the filtration unit 5 with microfiltration membrane and filtered, trapped fluid turns back in fermentor tank 3, filtered solution enters in concentration basin 6, and degerming rate reaches 96%; Wherein, microfiltration membrane is cellulose acetate film, and aperture is 1.2 μ m, and micro-filtration membrane module is plate-type flat facial mask, and filtration temperature is 33 DEG C, and pressure is 0.2MPa.The fermented liquid being trapped turns back in fermentor tank 3, and filtered solution enters concentration basin 6, and degerming rate reaches 96%.
3) ultra-filtration membrane and nanofiltration membrane are filtered: when the filtered solution volume after the degerming in concentration basin 6 reach fermentor tank volume 55% time, open the 3rd pump 7, the 3rd pump 7 flow velocitys are 10mL/min, and filtered solution is pumped into ultrafiltration instrument 8, and ultra-filtration membrane is polyvinylidene fluoride film, molecular weight cut-off is 50KD, hyperfiltration membrane assembly is rolling, and filtration temperature is 30 DEG C, and pressure is 0.5MPa, its trapped fluid turns back in fermentor tank 3, and filtered solution continues to filter, concentrate through nanofiltration instrument 9.Wherein, nanofiltration membrane is hollow fiber composite membrane, and molecular weight cut-off is 1500, and nanofiltration membrane component is rolling, and filtration temperature is 40 DEG C, pressure 0.5MPa.Trapped fluid after nanofiltration returns to concentration basin 6, does not return in fermentor tank 3 containing macromolecular filtered solution.
4) feed supplement bottle 1 is equipped with the fresh culture of sterilizing, pumps in fermentor tank, for the metabolism and growth of cell provides necessary nutrition by the first pump 2 with the speed of 1.0mL/L.Fresh culture pH value is 7.5, and at 115 DEG C sterilizing 30min; Fresh culture based component is: yeast powder 50g/L, W-Gum 8g/L, SODIUM PHOSPHATE, MONOBASIC 2g/L, Sodium phosphate dibasic 1.5g/L, MgSO 47H 2o3g/L, FeSO 47H 2o0.3g/L, CaCl 25g/L, MnCl 20.4g/L and glucose 15g/L.
5) trapped fluid in concentration basin 6 is concentrated, concentrated volume reach fermentor tank volume 10% time, then the mixture that adds methyl alcohol and acetic acid extracts (methyl alcohol and acetic acid volume ratio are 7:1); Soln using rotary evaporating device evaporation after extraction, obtains epothilone B after evaporate to dryness solution.
This method is produced epothilone B, and after fermentation ends, the accumulation volume of epothilone B reaches 48mg/L, and compared with not adopting batch fermentation of separation coupling, output has improved 60%.
The invention discloses a kind of method that epothilone B is produced in microorganism fermentation and membrane separation technique coupling.The present invention, mainly for the increase due to product epothilone B in fermenting process, can produce toxicity and feedback inhibition to cell, affects the thalli growth in fermenting process, proposes the method for separate fermentation coupling; Its step comprises, fermentation culture, and micro-filtrate membrane filtration, ultrafiltration and nanofiltration concentrate and feed supplement.The method makes fermented liquid product epothilone B concentration be reduced to lower level, has slowed down strain growth restraining effect, has improved the output of epothilone B, has reduced production cost, and is conducive to the aftertreatment of fermented liquid and refining of product.Make large-scale production become possibility, there is good suitability for industrialized production prospect.

Claims (10)

1. the method based on microorganism fermentation and membrane separation technique coupling production epothilone B, is characterized in that comprising the following steps:
1) fermentation culture: in 1L inoculation 10～100mg bacterial classification ratio, sorangium cellulosum is inoculated in fermentor tank and is fermented;
2) micro-filtrate membrane filtration: in fermenting process in the time that the output of epothilone B reaches 0.1mg/L, the fermented liquid in fermentor tank is filtered by the micro-filtration with microfiltration membrane, the fermented liquid of being held back by microfiltration membrane turns back in fermentor tank, continues fermentation, and filtered solution enters concentration basin;
3) ultra-filtration membrane and nanofiltration membrane are filtered: when the liquid bulk in concentration basin accumulate fermentor tank volume 50%～60% time, liquid in concentration basin is filtered by the ultrafiltration instrument with ultra-filtration membrane with the nanofiltration instrument of nanofiltration membrane, turn back in fermentor tank through the trapped fluid of ultra-filtration membrane, filtered solution continues to filter through nanofiltration membrane; Trapped fluid after nanofiltration membrane returns to concentration basin, and filtered solution turns back in fermentor tank;
4) the concentrated of epothilone B slightly carried: the liquid in concentration basin is concentrated, extracted, obtain epothilone B.
2. a kind of method based on microorganism fermentation and membrane separation technique coupling production epothilone B as claimed in claim 1, is characterized in that, described sorangium cellulosum is ATCC25532 or ATCC25569.
3. a kind of method of producing epothilone B based on microorganism fermentation and membrane separation technique coupling as claimed in claim 1, is characterized in that described step 1) in the pH value of fermention medium of fermentation employing be 7.2～7.5, and at 115 DEG C sterilizing 30min; Fermentation culture based component is yeast powder 20～50g/L, W-Gum 5～10g/L, SODIUM PHOSPHATE, MONOBASIC 1～2g/L, Sodium phosphate dibasic 1～2g/L, MgSO 47H 2o2～5g/L, FeSO 47H 2o0.1～0.5g/L, CaCl 22～5g/L, MnCl 20.1～0.5g/L and glucose 10～15g/L; Fermentation condition is: temperature is 28～32 DEG C, and mixing speed is 100～150r/min, and air flow is 0.3～0.6vvm.
4. a kind of method based on microorganism fermentation and membrane separation technique coupling production epothilone B as claimed in claim 3, is characterized in that, the fresh culture identical with fermentation culture based component is pumped in fermentor tank, carries out feed supplement during the fermentation; Liquid amount in fermentor tank is 60～75%.
5. a kind of method based on microorganism fermentation and membrane separation technique coupling production epothilone B as claimed in claim 1, it is characterized in that, hyperfiltration membrane assembly in micro-filtration membrane module, ultrafiltration instrument in described micro-filtration is plate-type flat facial mask, tubular type, rolling or tubular fibre formula, and the nanofiltration membrane component in nanofiltration instrument is rolling.
6. a kind of method based on microorganism fermentation and membrane separation technique coupling production epothilone B as claimed in claim 1, is characterized in that, described microfiltration membrane is cellulose acetate film, poly (ether sulfone) film or ceramic membrane, and aperture is 0.8 μ m, 1.0 μ m or 1.2 μ m; Ultra-filtration membrane is polyvinylidene fluoride film, hollow-fibre membrane or polyacrylonitrile film, and the molecular weight that dams is 1-150KD; Nanofiltration membrane is hollow fiber composite membrane, and the molecular weight that dams is 200～2000.
7. a kind of method based on microorganism fermentation and membrane separation technique coupling production epothilone B as described in claim 1 or 6, it is characterized in that, described step 2) in by the condition of filtering with the micro-filtration of microfiltration membrane be: temperature is 30～40 DEG C, and pressure is 0.2～0.5MPa.
8. a kind of method based on microorganism fermentation and membrane separation technique coupling production epothilone B as described in claim 1 or 6, it is characterized in that, described step 3) in ultrafiltration instrument by with the ultra-filtration membrane operational condition of filtering be: temperature is 30～40 DEG C, and pressure is 0.2～0.5MPa.
9. a kind of method based on microorganism fermentation and membrane separation technique coupling production epothilone B as described in claim 1 or 6, it is characterized in that, described step 3) in nanofiltration instrument by with the nanofiltration membrane condition of filtering be: temperature is 30～40 DEG C, and pressure is 0.2～0.5MPa.
10. a kind of method based on microorganism fermentation and membrane separation technique coupling production epothilone B as claimed in claim 1, it is characterized in that, described step 4) in extraction be to adopt the mixture of methyl alcohol and acetic acid to carry out, and in the mixture of methyl alcohol and acetic acid, the volume ratio of methyl alcohol and acetic acid is 7:3～9:1.
CN201410165832.9A 2014-04-23 2014-04-23 Method for producing epothilone B based on coupling of microbial fermentation and membrane separation techniques Pending CN103937851A (en)

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CN106834377A (en) * 2017-03-07 2017-06-13 鲁南制药集团股份有限公司 A kind of method for producing epothilone B
CN112481315A (en) * 2019-09-11 2021-03-12 吉林中粮生化有限公司 Method for preparing citric acid by continuous liquid change fermentation
CN113122443A (en) * 2021-04-23 2021-07-16 徐州生物工程职业技术学院 Energy-saving fermentation device for producing polypeptide drugs

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106834377A (en) * 2017-03-07 2017-06-13 鲁南制药集团股份有限公司 A kind of method for producing epothilone B
CN106834377B (en) * 2017-03-07 2020-05-05 鲁南制药集团股份有限公司 Method for producing epothilone B
CN112481315A (en) * 2019-09-11 2021-03-12 吉林中粮生化有限公司 Method for preparing citric acid by continuous liquid change fermentation
CN113122443A (en) * 2021-04-23 2021-07-16 徐州生物工程职业技术学院 Energy-saving fermentation device for producing polypeptide drugs
CN113122443B (en) * 2021-04-23 2021-09-10 徐州生物工程职业技术学院 Energy-saving fermentation device for producing polypeptide drugs

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Application publication date: 20140723