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

A microbial fermentation and epothilone technology, applied in the field of bioengineering, can solve the problems of stagnation or even decline in epothilone accumulation, decline in epothilone synthetase activity, and difficulty in further increasing production, so as to shorten production time , Operating conditions are mild, reducing the effect of inhibition

Inactive Publication Date: 2014-07-23
SHAANXI UNIV OF SCI & TECH
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  • Abstract
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Problems solved by technology

The main steps of small-scale preparation in the laboratory include: microbial fermentation, macroporous adsorption resin adsorption, analysis and separation, and drying to obtain the crude product. According to this method, epothilones accumulate continuously, the concentration of the fermentation liquid increases continuously, and the dissolved oxygen drops sharply. , the bacteria died, resulting in the decrease of the activity of epothilone synthase, the accumulation of epothilone in the fermentation broth stagnates or even declines, and the yield is difficult to further increase
And the operation of each unit in the laboratory production and preparation process is relatively independent, the production time is prolonged, the production efficiency is reduced, and the cost is increased
Membrane separation technology has not yet been applied to the production of epothilone

Method used

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

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Experimental program
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Effect test

Embodiment 1

[0029] see figure 1 , the present invention comprises the following steps:

[0030] 1) Fermentation culture: Inoculate 1 L of 100 mg of bacterial species, inoculate Cystobacter celluli ATCC25532 in a 5 L fermenter, and the liquid filling amount is 60%. The fermentation medium is composed of yeast powder 20g / L, corn starch 5g / L, sodium dihydrogen phosphate 1g / L, disodium hydrogen phosphate 1g / L, MgSO 4 ·7H 2 O2g / L, FeSO 4 ·7H 2 O0.1g / L, CaCl 2 2g / L, MnCl 2 0.1g / L and glucose 10g / L. The pH of the fermentation medium was 7.2, and it was sterilized at 115 °C for 30 min. The fermentation conditions in the fermenter 3 were as follows: the temperature was 30° C., the stirring speed was 100 r / min, and the ventilation rate was 0.3 vvm.

[0031] 2) Microfiltration membrane filtration: After the fermentation started, epothilone B continued to accumulate, and the concentration of the fermentation broth continued to increase. When the output of epothilone B reached 0.1 mg / L, the se...

Embodiment 2

[0037] see figure 1 , the present invention comprises the following steps:

[0038] 1) Same as Example 1.

[0039] 2) Microfiltration membrane filtration: After the fermentation started, epothilone B continued to accumulate, and the concentration of the fermentation broth continued to increase. When the output of epothilone B reached 0.1 mg / L, the second pump 4 was started, and the concentration was 10 mL / min. The flow rate of the partial fermentation culture liquid is pumped into the filter device 5 with microfiltration membrane for filtration, the retained fermentation liquid is returned to the fermentation tank 3, and the filtrate enters the concentration tank 6, and the sterilization rate reaches 96%; Among them, the microfiltration membrane is a polyethersulfone membrane with a pore size of 1.0 μm, the microfiltration membrane component is a plate-type flat membrane, the filtration temperature is 37° C., and the pressure is 0.5 MPa.

[0040] 3) with embodiment 1;

[00...

Embodiment 3

[0045] 1) with embodiment 1;

[0046] 2) with embodiment 1;

[0047] 3) ultrafiltration membrane and nanofiltration membrane filtration and concentration: when the filtrate volume after the sterilization in the concentration tank reaches 55% of the fermentor volume, the third pump 7 is opened, and the filtrate pump is pumped with the flow velocity of 10mL / min Enter into the ultrafiltration apparatus 8 with ultrafiltration membrane, and its retentate is returned to the fermentor 3, the filtrate continues to be filtered through the nanofiltration apparatus 9 with nanofiltration membrane, and the retentate after filtration returns to the concentration tank 6. The filtrate without macromolecules is returned to the fermentation tank 3; wherein, the ultrafiltration membrane is a hollow fiber membrane with a molecular weight cut-off of 150KD, the ultrafiltration membrane module is a tubular type, the filtration temperature is 37°C, and the pressure is 0.5MPa. The nanofiltration memb...

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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

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for producing epothilone B based on the coupling of microbial fermentation and membrane separation technology. Background technique [0002] Epothilones, a class of secondary metabolites produced by the myxobacteria Sorangium cellulosum, were first reported in 1993 by G.Hö,fle et al. of the German National Center for Biotechnology (GBF). Its relative molecular mass is 585. It has the functions of tubulin polymerization and microtubule stabilization, and has very high activity on cancer cells that are resistant to paclitaxel and other anticancer drugs; it has strong drug resistance and is easy to prepare and use, so epothilone As a potential new anti-cancer drug, it has aroused the research enthusiasm of biologists and pharmacists all over the world. One epothilone drug (BMS-247550, Ixabepilone, for the treatment of ovarian cancer) is currently on the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/18C07D493/04C12R1/01
Inventor 龚国利马利云
Owner SHAANXI UNIV OF SCI & TECH
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