CN101386874B - Two-liquid phase fermentation method for improving paclitaxel yield from fungi - Google Patents
Two-liquid phase fermentation method for improving paclitaxel yield from fungi Download PDFInfo
- Publication number
- CN101386874B CN101386874B CN2008101524992A CN200810152499A CN101386874B CN 101386874 B CN101386874 B CN 101386874B CN 2008101524992 A CN2008101524992 A CN 2008101524992A CN 200810152499 A CN200810152499 A CN 200810152499A CN 101386874 B CN101386874 B CN 101386874B
- Authority
- CN
- China
- Prior art keywords
- taxol
- fermentation
- substratum
- fungi
- paclitaxel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a method for improving the yield of taxol produced from fungi by two-liquid phase fermentation, which comprises the following steps: a fungus Pestalotiopsis microspora NK17 used to produce the taxol is cultured in a culture medium; when a thallus grows to a plateau phase, an organic solvent is added to continue the culture to ensure that the taxol is more produced and gathered in strain cells and the culture medium; and the taxol is recovered from the cells and culture medium and is purified. The method has high taxol yield, low production cost and short fermentation period, and is applied to industrial production; and the yield of the taxol can be trebled.
Description
[technical field]: the invention belongs to biotechnology and biological pharmacy technical field.Be specifically related to a kind of biliquid phase fermentation method that improves the paclitaxel produced productive rate of fungi.
[background technology]: taxol (paclitaxel, trade(brand)name Taxol) is a kind of natural cancer therapy drug of complexity, belong to diterpene alkaloid, its basic structure by Baccatine III (baccatin III) be connected its 13 carbon on a phenylalanine derivative constitute.
Because the market supply of taxol is limited by the restriction of raw material sources and technology of preparing two aspects, makes that the medicine high price is expensive.For this reason, new better raw material and method are being sought always by pharmaceutical industry and academia, replace existing production technology, with the output that improves taxol, satisfy clinical demand.The most significant progress that obtained in surplus past ten year is to have found to produce taxols with some fungies of plant symbiosis such as Ramulus et folium taxi cuspidatae, and its chemical structure and biological activity and Ramulus et folium taxi cuspidatae paclitaxel produced identical.
1993, Stierle etc. are separated to 1 strain endogenetic fungus-An Delie Japanese yew bacterium (Taxomyce sandreanae) from the phloem of Pacific Ocean Ramulus et folium taxi cuspidatae, through cultivating, adopt methods such as mass spectrum, chromatogram (TLC/HPLC) and radio chemistry mark, find to have taxol in the fermented liquid of this bacterium, although output is lower, every liter of fermented liquid contains taxol 24-50ng.Confirm also that simultaneously these fungi taxols and taxus brevifolia alcohol structure and character are just the same.Subsequently, many people are separated to different paclitaxel produced endosymbiosis fungies.China has also found multiple paclitaxel produced endosymbiosis fungi within the border.According to statistics, the generation taxol fungal species of having reported surpasses 30 kinds both at home and abroad, and these fungi overwhelming majority are ascomycetes or imperfect fungi, and all are the endosymbiosis fungies.Because the content of taxol is very low in these fungus fermentation products at present, can not reach the level that industrial fermentation is produced, therefore can not satisfy the demand in market far away.This laboratory is through a large amount of for many years further investigations, find and cultivated a kind of saprophytic fungus-plan dish stey Pestalotiopsis microspora NK-17 (culture presevation CGMCC2694), this bacterium taxol output is higher, fermentation condition is controlled easily, culture cycle is short, is the dominant bacteria that a strain industrial fermentation is produced taxol.
In the fermentation industry, except that single liquid phase fermentation (as amino acid fermentation, fermentation using enzyme, organic acid fermentation etc.) occupies the position of the utmost importance, biliquid phase fermentation system is being also to occupy critical positions in the fermentation (as tridecanyldicarboxylic acid production) of substrate and the bio-pharmaceuticals with the oil.At present, biliquid phase fermentation system has three classes in the fermentative production, the first kind be commercial be the fermentation system of carbon source with alkane or grease; Second class is the oxygen carrier fermentation system, soon oxygen is had higher solubility, but water-fast organic solvent (being called oxygen carrier) joins in the aerobic fermentation system oxygen transmission speed of quickening fermentation system; The 3rd class began one's study in recent years, in above-mentioned two class biliquid phase fermentation systems, introduce a kind of solid carrier (or emulsifying agent), the dual adsorptive power very strong and water and oil of utilizing this carrier to have increases the contact area between the biliquid phase, thereby improves mass transfer and fermentation production rate.
It is a kind of that the present invention promptly provides, and utilizes first kind biliquid phase fermentation system to improve the method that fungi plan dish stey is produced taxol.
[summary of the invention]: the objective of the invention is to overcome prior art above shortcomings and satisfy the demand of market, provide a kind of and utilize biliquid to ferment mutually to improve the paclitaxel produced method of fungi to taxol.
A kind of bacterial classification that can produce taxol provided by the invention is plan dish stey (Pestalotiopsis microspora) NK17 (culture presevation CGMCC2694).
Of the present invention utilize biliquid to ferment mutually to improve the paclitaxel produced method of fungi be: in substratum, cultivate plan dish stey Pestalotiopsis microspora NK17, when thalli growth reaches plateau, adding organic solvent continues to cultivate, so that produce in described microorganism cells and in the described substratum and the gathering taxol, and in described cell and the substratum, reclaim and purification of paclitaxel.
Described substratum is the PDB substratum, and it consists of g/L: potato 180-220g, sucrose 20g, natural pH.
Described yeast culture is the fermentation culture of carrying out in the 500mL-1L triangular flask, under the aerobic condition, vaccination ways adopts block mycelial vaccination ways, and culture temperature is 28-30 ℃, and the fermentation initial pH value is PDB substratum nature pH,, the cultivation rotating speed is 180-220rpm.
The organic solvent of selecting to add in continuing to cultivate is methyl alcohol, ethyl acetate or trichloromethane, and the scope of volumetric concentration is: 0.5%-2%.
Above-described cultivation can be carried out under the conventional or known condition of this area.Incubation time is decided according to culture condition, and time range is 6-10 days, and thalli growth enters plateau.
In order to isolate taxol, can use the conventional or known any separation method that is used for separating metabolite in this area from the substratum of microorganism from substratum.For example, mycelium can be separated from fermented liquid by centrifugal or filtration, and taxol can not extract from filtrate with the organic solvent that water mixes with one or more.On the other hand, the taxol that is comprised in the isolated mycelium can extract from mycelium by for example using a kind of solvent (for example methyl alcohol).Gained taxol crude product can use this area conventional or known purification process purifying, for example chromatography.
The method of extraction and purification of paclitaxel may further comprise the steps from described cell and substratum:
(1) described culturing process gained liquid is isolated fermentation thalline and fermented supernatant fluid;
(2) with organic solvent extraction gained fermentation thalline, extracting solution concentrates with fermented supernatant fluid of last step;
(3) with the method purifying of (2) step gains, get the product taxol with chromatogram purification.
In described (1) step, can separate for example centrifugal or filtration by this area routine or known method with the gained fermented liquid.
Can use organic solvent extraction again with after the dry pulverizing of gained zymophyte soma in described (2) step.Use saturated Na
2CO
3The pH value that solution is regulated extracting solution and fermented supernatant fluid is 6.0-8.0.Described drying can be carried out according to this area routine or known method, for example vacuum freeze-drying method.The described organic solvent that the extraction taxol is used from the fermentation thalline can be conventional or known solvent, for example a methyl alcohol of this area.Described concentration method can carry out according to this area routine or known method, for example concentrating under reduced pressure.
Chromatogram purification in described (3) step can carry out according to this area routine or known method, and chromatographic column filler can be used C18, and moving phase can be used methyl alcohol/H
2O also can use more than one chromatographic column.
Advantage of the present invention and positively effect:
Adopt the method for biliquid phase fermentative production taxol of the present invention, productive rate can reach 551.72 μ g/L, and productive rate is higher than existing tissue culture method and microbe fermentation method more than 1 times.The present technique cycle is short, and cost is low, and the productive rate height is applied to suitability for industrialized production, can solve the technological problems of taxol, has promptly protected natural resources, can satisfy the needs of clinical application again.
[description of drawings]:
Fig. 1 is the influence of the biliquid phase system of different sorts and proportioning among the present invention to plan dish stey NK17 bacterial strain taxol output.
The paclitaxel produced new bacterial strain of energy provided by the invention is a strain saprophytic fungus plan dish stey, be preserved in Chinese common micro-organisms culture presevation administrative center on October 8th, 2008, preservation address: No. 3 Institute of Microorganism, Academia Sinica in A, DaTun Road, Chaoyang District, BeiJing City, culture presevation CGMCC2694, classification name: Pestalotiopsis microsporaNK17.
[embodiment]:
Below will the invention will be further described by embodiment, these descriptions are not that content of the present invention is done further to limit.One skilled in the art will understand that invention technical characterictic being equal to of being done replaced or corresponding the improvement, still belong within protection scope of the present invention.
The fermentation culture of embodiment 1, paclitaxel produced bacterial strain NK17
Cut a fritter NK17 mycelium with scalpel and insert in the PDB substratum of the bacterium of having gone out, PDB consists of (g/L): potato 180-220g (as 180,200 or 220g), sucrose 20g, natural pH.Yeast culture is in the 500mL-1L triangular flask, and the fermentation culture of carrying out under the aerobic condition, culture temperature are 28-30 ℃, and the fermentation initial pH value is PDB substratum nature pH, the cultivation rotating speed be 180-220rpm (as 180,200 or 220rpm).Treat that thalli growth reaches (the 7th day) after plateau, selecting to add organic solvent is methyl alcohol, ethyl acetate or trichloromethane, and the scope of volumetric concentration is: 0.5%-2%.
The concrete organic solvent of selecting to add is 1% methyl alcohol, or 0.5% ethyl acetate, or 1% ethyl acetate, or 2% ethyl acetate, or 0.5% trichloromethane, or 1% trichloromethane.Continue then to cultivate 3～4 days.
Embodiment 2, paclitaxel produced bacterial strain NK17 tunning pre-treatment process
To go up routine tunning and carry out suction filtration, and make mycelia separate with fermented supernatant fluid; With gained mycelia lyophilize behind the suction filtration, used the methyl alcohol extracting 1-2 days after the liquid nitrogen grinding smudge cells; With extract with carry out suction filtration once more after before fermented supernatant fluid mixes, disgorging, and regulate pH to 7.0 (pH value be 6.0 or 8.0 all can, preferably pH is 7.0); Slightly carried product after using rotatory evaporator that gained filtrate of last step is concentrated, carried out the separation and the purifying of next step product.
The separation of embodiment 3, product and purifying
(4.6 * 250mm, Kromasil) NK of embodiment 2 gained is slightly carried product and carry out purifying and analysis, chromatographic condition: detect wavelength 227nm, flow velocity 1mL/min, column temperature are room temperatures, and moving phase is methyl alcohol: water=7:3 to select analysis mode C-18ODS chromatographic column.Sharp side according to the taxol in each sample that draws in the HPLC process is long-pending, and the normalized form of the calculating content of taxol that the utilization laboratory has obtained calculates yield.As can be seen from Figure 1, when adding 1% methyl alcohol, the content of taxol is greatly improved.Compare with control group, increased by one times nearly.And add 2% ethyl acetate, and or 0.5% trichloromethane, or during 1% trichloromethane, the output of taxol also has raising in various degree.
Claims (7)
1. biliquid phase fermentation method that improves the paclitaxel produced productive rate of fungi, it is characterized in that: cultivating culture presevation in substratum number is plan dish stey (Pestalotiopsis microspora) NK17 of CGMCC No.2694, when thalli growth reaches plateau, adding volumetric concentration is the organic solvent continuation cultivation of 1% methyl alcohol, 2% ethyl acetate or 0.5%-1% chloroform, so that produce in described microorganism cells and in the described substratum and the gathering taxol, and in described cell and the substratum, reclaim and purification of paclitaxel.
2. method according to claim 1 is characterized in that described substratum is the PDB substratum of nature pH, and it consists of g/L: potato 180-220g, sucrose 20g.
3. method according to claim 1 is characterized in that described cultivation is under aerobic conditions to carry out, and temperature is 28-30 ℃, and the fermentation initial pH value is PDB substratum nature pH, and the cultivation rotating speed is 180-220rpm.
4. method according to claim 1 is characterized in that thalline is a fermentation culture in the 500mL-1L triangular flask, and vaccination ways adopts block mycelial vaccination ways.
5. according to any described method in the claim 1 to 4, it is characterized in that the step of described recovery and purification of paclitaxel comprises:
(1) isolates fermentation thalline and fermented supernatant fluid from the gained nutrient solution;
(2) with organic solvent extraction gained fermentation thalline, extracting solution concentrates with the fermented supernatant fluid in last step;
(3) with the method purifying of (2) step gains, get the product taxol with chromatogram purification.
6. method according to claim 5 is characterized in that using saturated Na before (2) step concentrated
2CO
3The pH value that solution is regulated extracting solution and fermented supernatant fluid is 6.0-8.0.
7. method according to claim 5 is characterized in that: in the described chromatogram purification method of (3) step, use one or more chromatographic column, filler is C18, and moving phase is methyl alcohol/H
2O.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101524992A CN101386874B (en) | 2008-10-28 | 2008-10-28 | Two-liquid phase fermentation method for improving paclitaxel yield from fungi |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101524992A CN101386874B (en) | 2008-10-28 | 2008-10-28 | Two-liquid phase fermentation method for improving paclitaxel yield from fungi |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101386874A CN101386874A (en) | 2009-03-18 |
| CN101386874B true CN101386874B (en) | 2011-05-11 |
Family
ID=40476544
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008101524992A Expired - Fee Related CN101386874B (en) | 2008-10-28 | 2008-10-28 | Two-liquid phase fermentation method for improving paclitaxel yield from fungi |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101386874B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506021B (en) * | 2015-12-31 | 2019-05-24 | 浙江省林业科学研究院 | One kind composition of culture containing taxol and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1511943A (en) * | 2002-12-30 | 2004-07-14 | 黑龙江大学 | Engineering strain and its preparing method, and method for preparing taxol thereof |
| CN1624103A (en) * | 2004-11-13 | 2005-06-08 | 西南师范大学 | Process for raising producing rate of taxad alcohol of Taxus chinensis endogeny fungus fermenting material |
-
2008
- 2008-10-28 CN CN2008101524992A patent/CN101386874B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1511943A (en) * | 2002-12-30 | 2004-07-14 | 黑龙江大学 | Engineering strain and its preparing method, and method for preparing taxol thereof |
| CN1624103A (en) * | 2004-11-13 | 2005-06-08 | 西南师范大学 | Process for raising producing rate of taxad alcohol of Taxus chinensis endogeny fungus fermenting material |
Non-Patent Citations (4)
| Title |
|---|
| Gary Strobel,et al..Taxol from Pestalotiopsis microspora, entophytic fungus of Taxus wallachiana.《Microbiology》.1996,第142卷第435-440页. * |
| GaryStrobel et al..Taxol from Pestalotiopsis microspora |
| 吴兆亮等.南方红豆杉细胞双液相培养中强化紫杉醇生产的研究.《植物学报》.1999,第41卷(第10期),第1108-1113页. * |
| 贾士儒等.双液相发酵的进展.《食口与发酵工业》.1996,(第5期),第58-62页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101386874A (en) | 2009-03-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lin et al. | Evaluation of different agricultural wastes for the production of fruiting bodies and bioactive compounds by medicinal mushroom Cordyceps militaris | |
| CN104342390B (en) | A kind of Sinorhizobium meliloti strain and combinations thereof and application | |
| CN103911293B (en) | The endogenetic fungus that one plant height is paclitaxel produced and apply this bacterial strain and produce the method for taxol | |
| CN102807956B (en) | Ceriporia lacerata strain and application thereof | |
| CN103416223A (en) | Method for improving cordycepin output in cordyceps militaris fermentation broth | |
| CN106010980B (en) | A kind of endogenetic fungus Brazil class shell roundlet spore bacterial strain and its application | |
| CN103255194B (en) | Method for raising conversion rate of 15 alpha, 17 alpha-epoxyprogesterone | |
| CN109182147A (en) | A kind of mould and its method for producing fumidil | |
| CN102061278B (en) | Methylovorus sp. MP688 and application thereof | |
| CN100999750B (en) | Process of synthesizing gastrodiacin by microorganism cell biological transferring parhydroxy benzyl methylol | |
| CN102485879A (en) | Fermentation medium used for producing WF 11899A | |
| CN102161973B (en) | Method for preparing homoharringtonine and special strain | |
| CN100516195C (en) | New strain and method of producing pacilitaxel using said strain | |
| CN102417883A (en) | Screening of new strain capable of producing camptothecin and method for preparing camptothecin | |
| CN103146614B (en) | Camptotheca endophytic bacterium LY214 for producing camptothecin and application thereof | |
| CN101386874B (en) | Two-liquid phase fermentation method for improving paclitaxel yield from fungi | |
| CN101386819B (en) | Saprophytic fungus for producing paclitaxel and method for producing paclitaxel using thereof | |
| CN113337432B (en) | Methylophilus for producing pyrroloquinoline quinone and application thereof | |
| CN103725728B (en) | The preparation method of a kind of Bacillamide compound and Bacillamide precursor | |
| CN104250621A (en) | Sea streptomyces cinereoruber and method of applying same to prepare aurone compound | |
| CN102757443A (en) | Sulfur-substituted podophyllum derivative and bioconversion, separation and purification method thereof | |
| CN105316238B (en) | A method of the culture screening taxol-producing fungi from Chinese yew | |
| CN102584615A (en) | Alkaloid compound as well as preparation method and application thereof | |
| CN103074236A (en) | Camptotheca endophytic fungi for producing camptothecin and application thereof | |
| CN101418341A (en) | Method for quickly screening taxol-producing endophytic fungi |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110511 Termination date: 20171028 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |