CN101892162A - New method for producing taxol by fermenting taxus chinensis branches and leaves by using taxol producing strains - Google Patents
New method for producing taxol by fermenting taxus chinensis branches and leaves by using taxol producing strains Download PDFInfo
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- CN101892162A CN101892162A CN2010101709892A CN201010170989A CN101892162A CN 101892162 A CN101892162 A CN 101892162A CN 2010101709892 A CN2010101709892 A CN 2010101709892A CN 201010170989 A CN201010170989 A CN 201010170989A CN 101892162 A CN101892162 A CN 101892162A
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Abstract
The invention discloses a new method for producing taxol by fermenting southern taxus chinensis branches and leaves by using taxol producing strains. The method is characterized in that: the taxol producing strains are cultured in a solid MS culture medium and fermentation is performed in a liquid MS culture medium and the method comprises the following steps of: cleaning, dewaxing and sterilizing fresh taxus chinensis branches and leaves to obtain a main fermentation raw material, adding a liquid basic MS culture medium into fermentation strains for blending and then inoculating, performing fermentation under the condition of the temperature of 22+/-3 DEG C and extracting, purifying and drying after a fermentation process is finished so as to obtain a pasty taxol crude extract. The method for culturing the taxol producing strains is simple, and the method for producing the taxol has the advantages of high taxol precursor transformation efficiency in a taxus chinensis branch and leaf fermentation process and capability of controlling temperature to be not higher than 40 DEG C in an entire process, avoiding damage to taxol structure and enhancing taxol extraction rate.
Description
Technical field
The present invention relates to a kind of taxol and produce the isolation and selection method of bacterial strain and utilize this strain fermentation Ramulus et folium taxi cuspidatae to transform the taxol precursor, improve the Technology of taxol yield.
Background technology
Taxol is the secondary metabolite of a kind of complexity in the Chinese yew genus plants, by extracting or semisynthetic effective constituent in yew tree, bark or the needle, also be that understood at present only a kind of can promote microtubule polymerization and the stable medicine of polymerization microtubule.By the II-III clinical study, taxol mainly is applicable to ovarian cancer and mammary cancer, and lung cancer, large bowel cancer, melanoma, incidence cancer, lymphoma, brain tumor are also all had certain curative effect.Because its mechanism of action uniqueness is effective to a lot of resistance patients, become one of at present the most popular antitumor drug.
Ramulus et folium taxi cuspidatae is a kind of poky arbor, and the temperate zone that is distributed in the Northern Hemisphere is to the subtropical zone, and there are four kinds and a mutation (southerm yew) in China, but whole world total resources is but extremely limited, scattered being distributed in the wildwood.Present global taxol demand is tens of to hundreds of tons of pure product of paclitaxel in every year, and seeking new source becomes main research direction.
1, the different sites from Ramulus et folium taxi cuspidatae extracts taxol
Mainly extract and produce taxol by bark of Ramulus et folium taxi cuspidatae, but destroy huge to wild resource, people attempt to extract to enlarge the taxol source from other positions of Ramulus et folium taxi cuspidatae plant, Kelth Witherup has measured the content of taxol in 6 kinds of Ramulus et folium taxi cuspidatae and the stem, finds that the content of taxol in the needle is higher.
2, stock breeding, artificial culture
For remedying the in great shortage of natural resource, staff both domestic and external carries out Ramulus et folium taxi cuspidatae fine-variety breeding and cuttage and quick-propagation technical study, wherein country of University of Pennsylvania natural product technique center has filtered out the plant of high content of taxol for artificial culture, USDA and University of Mississippi's associating cultivating Chinese yew scale reach millions of strains, can gather in 3～5 years.
3, chemosynthesis taxol
1. complete synthesis taxol
The chemistry professor Hao Dun of the upright university of Fla. is a parent material with camphor, adopts linear synthesis method, and it is complete synthesis to finish taxol in December, 1993; Earlier synthetic two six-rings of Scipps institute 1992 annual reports, it being formed continuously with the octatomic ring is the intermediate at center again, and then synthetic tetra-atomic ring, thereby finishes the complete synthesis of taxol.But two kinds of complete synthesis approach all comprise the chemical reaction complex process in 25～30 steps, apart from suitability for industrialized production very big distance are arranged still.
2. semi-synthetic taxol
1988, French Denis reported first from the needle of european yew, obtain 10-acetyl Baccatine III (baccatin-III), as raw material semi-synthetic taxol and homologue texotere thereof.U.S. Rhone-Poulene Rorer chemical company has also realized the semi-synthetic of taxol subsequently, and has applied for baccatin-III being the patent of the semi-synthetic taxol of raw material.U.S. BMS corporation plan stops the activity in production of extraction taxol from bark after obtaining drugs approved by FDA, utilize semi-synthesis method to carry out the suitability for industrialized production taxol.
4, utilize the plant cell engineering method to produce taxol
Utilize the culture plant cell method to produce taxol, this method once once had been a very attractive direction in the various research directions, also be in the taxol symposial held of nineteen ninety U.S. NCI, obtain one of direction of NCI investment, but do not have breakthrough over 10 years.
5, paclitaxel analogs
At present the countries in the world scientist separates from Chinese yew genus plants and obtains more than 200 kind of compound, all has the basic framework of Taxan tricyclic diterpene, partly has anti-tumor activity.
Analysis-by-synthesis, at present both at home and abroad a lot of to the research direction of taxol, but there is not the carried out suitability for industrialized production of moulding, major cause is the taxus resource deficiency, the seeds poor growth of cultivation can not provide enough raw materials in the short period of time; Taxol structural formula complexity, the suitability for industrialized production that is difficult to carry out chemosynthesis, cell culture processes is because fermentation period is long and high yielding cell sarain seed selection length consuming time, and pilot scale is gone into operation all certain difficulty.So Chinese scholars begins to turn to the fungi fermentation method to produce taxol, the key issue that runs at present is that the raising that taxol produces content of taxol in the separation of bacterium and the fermented liquid becomes technical bottleneck.
Summary of the invention
For carrying out the suitability for industrialized production of taxol, the present invention works out a kind of novel taxol production technique by production practice.May further comprise the steps:
1, taxol produces the seed selection of bacterium: adopt the fungi that separates from the southerm yew phloem, through ultraviolet mutagenesis, filter out taxol precursor activity of conversion is strong, rate of propagation is fast " WM20094001 " number bacterial strain as producing bacterial strain.
2, " WM20094001 " number bacterial strain slant culture based formulas is a solid MS substratum, and the shake-flask culture base is a liquid MS medium, and fermention medium is a liquid MS medium.
3, after the fermented liquid about taxol production bacterium fermentation 72h dilutes 5 times with the nutritive medium of developing voluntarily, admix Ramulus et folium taxi cuspidatae, about processing 120h, transform the taxol precursor under 20 ℃～25 ℃ conditions through pre-treatment.
4, Ramulus et folium taxi cuspidatae is handled and is adopted hot steam instantaneous surface dewaxing and sterilization, is tiled on the fermentation bed, adopts the mode of ventilating fermentation to ferment.
5, after the fermentation, mechanical stirring becomes pasty state, adds long-pending 95% ethanol of triploid, stirs, and leaves standstill lixiviate 30min, and extracting solution removes slag through centrifugal, in vacuum-drying below 40 ℃, obtains medicinal extract shape product.
Innovation of the present invention is embodied in the following aspects:
1. Technology innovation: utilize Technology of the present invention, the consistent lesser temps (below 40 ℃) that keeps does not destroy the taxol structure in the production process, improves yield, does not relate to step, less energy-consumption and three-waste free discharges such as raw material drying simultaneously.
2. original innovation: the taxol that selects is produced bacterium as unique production bacterial strain, and the taxol precursor that transforms in the Ramulus et folium taxi cuspidatae improves taxol yield, is that former research is never mentioned.
The present invention adopts isolating taxol generation bacterium in the southerm yew, is inoculated in the Ramulus et folium taxi cuspidatae material through dewaxing, disinfecting, and carries out heat-preservation fermentation, utilizes the taxol precursor biosynthesizing taxol in the Ramulus et folium taxi cuspidatae.Not only solved is the shortage of raw materials problem of the production taxol of raw material with the bark of Ramulus et folium taxi cuspidatae, the complex chemical reaction process control problem of having evaded chemical synthesis again, utilize the natural microbial of seed selection to ferment, the cell strain seed selection time is short, go into operation and go up scale easily, extraction process is simple, has thoroughly solved the various limiting factors of suitability for industrialized production.
Embodiment
By embodiment the present invention is further described
Embodiment one
1 material and instrument
The fresh branches and leaves raw material of southerm yew (Lu'an City grinds sincere agricultural science and technology limited liability company and provides), sodium hydroxide, hydrochloric acid, glucose, (NH
4)
2NO
3: KH
2PO
4: MgSO
4: FeSO
4Deng being homemade analytical pure, potato etc. are purchased from the local market.
Bechtop, constant incubator, shaking table, full-automatic in stainless steel fermentor tank (SGD1000) on probation, 5m
3Container for storing liquid, ventilating fermentation pond (1.2m * 0.8m * 8m), industrial whizzer, vacuum dryer, plate-and-frame filter press, alcohol recovery system (JS-500)
2 experimental techniques
2.1 taxol is produced bacterium and is cultivated
Produce bacterial strain: Alternaria tenuissima " WM20094001 " number bacterial strain, the fresh and alive southerm yew phloem that separates arrogant other mountain area growth by the Lu'an City Western Hills biotechnology scientific research personnel of limited liability company, through ultraviolet mutagenesis, the bacterial strain that taxol precursor activity is the strongest, rate of propagation is the fastest that filters out.
Inclined-plane seed culture medium: solid MS substratum.
Liquid seed culture medium: liquid MS medium.
Fermention medium: liquid MS medium.
Bacterium liquid is made: get the activatory slant strains, be inoculated in the seed culture medium, every shaking flask inoculation 1 ring, 150r/min on shaking table cultivates about 72h, as seed liquor for 25 ℃, inserting fermentor tank in 5% ratio ferments, 1.8L/min 25 ℃ of air flows cultivate about 72h, with 4 times of liquid basic MS culture medium dilutions, as inoculating bacterium liquid to fermentating liquid volume.
2.2 fermenting raw materials (the taxol precursor transforms in the Ramulus et folium taxi cuspidatae)
The fresh branches and leaves of Ramulus et folium taxi cuspidatae through 125 ℃ of steam treatment 1min about, after temperature is cooled to room temperature, be tiled on the fermentation bed in ventilating fermentation pond, evenly spray taxol production with inoculation bacterium liquid, handle 120h under 22 ℃ ± 3 ℃ conditions and finish fermenting process.
3. the Ramulus et folium taxi cuspidatae after will fermenting, machinery is smashed pulping, add 3 times to 95% ethanol of soup compound volume, stir, leave standstill the back layering, incline and supernatant liquor, the beds of precipitation adopt the plate-and-frame filter press press filtration, and filtrate merges the centrifugal impurity of removing in back with the supernatant liquor that inclines, and carries out low-temperature vacuum drying, obtain paste taxol crude product, reclaim ethanol and can be recycled.
3 conclusions
Utilize Alternaria tenuissima " WM20094001 " number bacterial strain, carry out the fresh branches and leaves of Ramulus et folium taxi cuspidatae by above-mentioned technical process and handle, the taxol of acquisition directly extracts the taxol yield than fresh branches and leaves and improves 1.2 times.
Experimental result proves; the taxol that adopts present method to produce; the working condition gentleness; little to the taxol structural impairment that contains in the Ramulus et folium taxi cuspidatae, farthest transformed the taxol precursor to the greatest extent simultaneously, saved resource; improved the taxol yield; and less energy consumption, pollution-free, help environment protection and wild taxus resource.
[special instruction]: above the preferred embodiments of the present invention mode has been done detailed description, but the invention is not restricted to above-mentioned embodiment, those of ordinary skill in the art can also make other equivalence and change and modify under the prerequisite that does not break away from aim of the present invention.
Claims (2)
1. the present invention relates to a kind of paclitaxel produced novel bacterial, it is characterized in that: described novel bacterial was the bacterial strain of separation screening from the wild southerm yew bark in this locality, and this bacterial strain was entrusted Chinese microorganism strain preservation center (CGMCC) to make patent and preserved in 2007.
2. paclitaxel produced novel bacterial fermentation Ramulus et folium taxi cuspidatae according to claim 1 is produced the taxol method, it is characterized in that:
(1) the strain fermentation substratum adopts liquid MS medium;
(2) Ramulus et folium taxi cuspidatae is handled: the fresh branches and leaves of Ramulus et folium taxi cuspidatae through 125 ℃ of steam treatment 1min about, after temperature is cooled to room temperature, be tiled on the fermentation bed in ventilating fermentation pond.
(3) fermentation culture: get the activatory slant strains, be inoculated in the liquid MS medium, every shaking flask inoculation 1 ring, 150r/min on shaking table, cultivate about 72h for 25 ℃, as seed liquor, inserting fermentor tank in 5% ratio ferments, 1.8L/min 25 ℃ of air flows cultivate about 72h, with 4 times of liquid basic MS culture medium dilutions to fermentating liquid volume,, evenly be sprayed on the Ramulus et folium taxi cuspidatae of handling well with inoculation bacterium liquid as taxol production, ventilating fermentation 120h finishes fermenting process under 22 ℃ ± 3 ℃ conditions.
(4) extraction of taxol: the Ramulus et folium taxi cuspidatae after will fermenting, machinery is smashed pulping, add 3 times to 95% ethanol of soup compound volume, stir, leave standstill the back layering, incline and supernatant liquor, the beds of precipitation adopt the plate-and-frame filter press press filtration, and filtrate merges the centrifugal impurity of removing in back with the supernatant liquor that inclines, and carries out low-temperature vacuum drying, obtain paste taxol crude product, reclaim ethanol and can be recycled.
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| CN2010101709892A CN101892162A (en) | 2010-04-24 | 2010-04-24 | New method for producing taxol by fermenting taxus chinensis branches and leaves by using taxol producing strains |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104605138A (en) * | 2013-11-01 | 2015-05-13 | 南京黑玉特种家禽养殖专业合作社 | Poultry feed additive containing fermented Chinese yew, and preparation method and applications thereof |
| CN113736835A (en) * | 2021-09-10 | 2021-12-03 | 陈开云 | Industrial preparation process for producing taxol by fermenting taxus chinensis endophytic fungi mutant |
| CN113980819A (en) * | 2021-10-29 | 2022-01-28 | 江苏师范大学 | Taxus chinensis needle endophytic fungi strain and application thereof |
-
2010
- 2010-04-24 CN CN2010101709892A patent/CN101892162A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104605138A (en) * | 2013-11-01 | 2015-05-13 | 南京黑玉特种家禽养殖专业合作社 | Poultry feed additive containing fermented Chinese yew, and preparation method and applications thereof |
| CN113736835A (en) * | 2021-09-10 | 2021-12-03 | 陈开云 | Industrial preparation process for producing taxol by fermenting taxus chinensis endophytic fungi mutant |
| CN113980819A (en) * | 2021-10-29 | 2022-01-28 | 江苏师范大学 | Taxus chinensis needle endophytic fungi strain and application thereof |
| CN113980819B (en) * | 2021-10-29 | 2023-11-14 | 江苏师范大学 | Taxus chinensis needle leaf endophytic fungus strain and application thereof |
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Application publication date: 20101124 |