CN1288953A - Method of producing taxad alkane by using Chinese yew cell - Google Patents
Method of producing taxad alkane by using Chinese yew cell Download PDFInfo
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- CN1288953A CN1288953A CN 00125305 CN00125305A CN1288953A CN 1288953 A CN1288953 A CN 1288953A CN 00125305 CN00125305 CN 00125305 CN 00125305 A CN00125305 A CN 00125305A CN 1288953 A CN1288953 A CN 1288953A
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Abstract
The present invention utilizes the suspension culture of Taxus Chinensis and uses the induction of inducing agent and combination of addition of supplemental sugar so as to greatly raise the production efficiency of yannan taxane (Tc), its maximum production efficiency can be up to 30.2 mg/(L.d), and its maximum yield can be up to 538.9 mg/L.
Description
The present invention relates to the production method of Taxan, relate in particular to the method for producing Taxan with plant cell culture technology.
TAXUYUNNANINE C (Taxuyunnanine C, Tc) belong to the tricyclic diterpene compounds, document with taxol: (1994) Taxanes from Taxus chinensis.Phytochemistry.38:667-670. has reported this compound, and its structural formula is as follows:
Taxan Tc (being called for short Tc) is presumably the intermediate metabolites in the taxol biosynthetic pathway, they can be used as the precursor of taxol biosynthesis or other useful matter, among the pharmaceutical use of itself being is also is being researched and developed (the strong Ph D dissertation of Wang Hong, East China University of Science, 1998).Utilize yew cell cultivation high yield Taxan to have potential commercial production value as the precursor of the semi-synthetic taxol of chemistry.
Since the clinical antitumour activity of taxol was found, taxol and relevant bearing taxanes were subjected to people and more and more pay close attention to.Up to the present, separated obtaining more than 100 kind of Taxan, and found that some taxaneses have the activity similar with taxol.U.S. food and FAD (FDA) can be used for treating the advanced ovarian cancer patient in official approval in the end of the year 1992 taxol.Up to the present, existing more than ten state approval taxol can be used for treatment for cancer.But whole world taxus resource is very limited, and very big to its demand.According to estimates, the whole world needs the taxol of 500kg could satisfy numerous cancer patientss'demand every year.If light extracts taxol from bark, the annual big tree that needs about ten thousand of felling 150-200.Even need the 50kg taxol to calculate every year by the whole world, whole world Chinese yew genus plants is also only enough cut down 10-15.So a large amount of felling will bring serious threat to retaining for a long time with the area distribution of Chinese yew genus plants, cause the very big destruction and the irreparable damage of species diversity and ecotope.So other source of research taxol has become the task of top priority.Compare patent with methods such as artificial culture Taxus plant or chemical complete synthesis taxols:the cell culture method of reports such as Japan Patent WO 95/14103, US Patent 5407816 produce the taxanes material because with short production cycle, the labor force economizes, cost is low and be subjected to advantages such as external environment influence is little to be considered to a kind of relatively effective means usually.Still there is the low problem of production efficiency at present but produce taxol, do not reach industrial production requirement with plant cell culture technology. derivant owing to its have can stimulating plant cell cometabolism ability be used to further improve the production capacity of plant cell, document: (1996) Methyl jasmonate-induced overproduction of paclitaxel and baccatin III inTaxus cell suspension cultures.Nat.Biotechnol.14:1129-1132. and (1999) The kineticsoftaxoid accumulationin cell suspension cultures of Taxus following elicitation withmethyl jasmonate.Biotechnol.Bioeng.62:97-105. have reported that jasmone acids substance enough promotes the generation of taxane substances. But the research that utilizes revulsion to improve TAXUYUNNANINE C (Tc) throughput yet there are no report.
The object of the present invention is to provide a kind of suspension culture of utilizing taxus chinensis cell (Taxus chinensis), induce and mend the novel method that sugar adds the production TAXUYUNNANINE C (Tc) combine by inductor, very improve the production efficiency of Tc significantly, overcome the defective of prior art.
Realize that technical scheme of the present invention comprises the steps:
(1) cultivation of taxus chinensis cell:
To from plant children stem explant induction callus, separate Chinese Ramulus et folium taxi cuspidatae (T.chinensis) cell that obtains, move into and adopt conventional method to carry out succeeding transfer culture in the liquid nutrient medium, this preparation process is a prior art, at document: in (1997) Enhanced production oftaxol in suspension cultures of Taxuschinensis by controlling inoculum size.19:353-355. document to the existing detailed description of this process; Substratum adopts the disclosed Murashige-Skoog of the document (MS) substratum, other adds the 0.1-1.0mg/L6-benzyladenine, the 0.05-0.5mg/L 2,4 dichlorophenoxyacetic acid, 0.05-0.5mg/L naphthylacetic acid, 20-500mg/L xitix and 10-50g/L sucrose.Went down to posterity once every 14 days, cultivate and use rotary shaking table, rotating speed is 60-200rpm, culture temperature 20-30 ℃, and dark the cultivation, inoculum size is the 20-200g/L weight in wet base.
(2) production of Tc:
The Chinese Ramulus et folium taxi cuspidatae wet cell that step (1) is obtained places the bottle that shakes that contains above-mentioned substratum secretly to cultivate (inoculum size is the 20-200g/L weight in wet base), rotary shaking speed is 60-200rpm, temperature 20-30 ℃, culture cycle is 12-25 days, from the culture that is obtained, adopt conventional method to collect Tc, Tc productivity reaches as high as 30.2mg/ (Ld), and production peak can reach 538.9mg/L.
In the shake-flask culture process, can utilize inductor to induce, cultivating the quick growth phase of cell in early stage, adding inductor dihydro methyl jasmonate (HMJA) or methyl jasmonate (MJA) ultimate density of inductor to the substratum is 1-1000 μ M;
Mend sugar when adding inductor, additional amount is 10-30g/L;
Said sugar is sucrose, glucose, fructose, lactose etc.
(3) measuring method of Tc
Take by weighing the 100mg stem cell, wear into fine powder, adding 2mL methyl alcohol soaked 3 days, get supernatant liquor after centrifugal and volatilize, add methylene dichloride and each 2mL of distilled water, fully concussion was left standstill several minutes after mixing, centrifuging and taking lower floor methylene dichloride phase, volatilize, add 1mL HPLC level dissolve with methanol, be used for the Tc efficient liquid phase chromatographic analysis.Employing is furnished with Waters 510 (Britain) the type high performance liquid chromatograph of Waters 486 Ultraviolet Detectors, and the detection wavelength is 227nm.The penta fluoro benzene silane post (PFP) that adopts U.S. Whatman company to produce.Moving phase is own nitrile: water=42: 58, flow velocity are 1mL/min.Each sample size is 20 μ L, and sample removes solid impurity with the 10000rpm centrifuging and taking before the sample introduction.
By above-mentioned disclosed technical scheme as seen, the present invention utilizes simultaneously and induces the throughput that promotes cell with feed supplement, the Tc output that obtains substantially exceeds the maximum of present bibliographical information, Tc productivity reaches as high as 30.2mg/ (Ld), production peak can reach 538.9mg/L, is a kind of production method that the Tc of prospects for commercial application is arranged very much.
Below will be further described related content of the present invention by embodiment.
Embodiment 1
The taxus chinensis cell that employing obtains from plant children stem explant induction callus.The 250mL shake-flask culture adopts the MS substratum, and other adds the 0.5mg/L 6-benzyladenine, 0.2mg/L 2,4 dichlorophenoxyacetic acid, 0.2mg/L naphthylacetic acid, 100mg/L xitix and 30g/L sucrose.
The 5g wet cell that vacuum filtration is obtained be inoculated into contain the above-mentioned substratum of 50mL shake in the bottle rotary shaking speed 110rpm, 25 ℃ of cultivations.Added in the 9th day MJA to ultimate density be 100 μ M, continue to be cultured to 21 days and finish fermentation, harvested cell and 40 ℃ of dryings.Cell density reached the highest (dry weight 19.2g/L) at the 12nd day, productive rate is 1.0g/ (Ld).In addition, stem cell also will carry out the assay determination of Tc, calculate the results are shown in following table (
*Represent maximum value).
| Incubation time (my god) | Tc content (mg/gDW) | Tc output (mg/L) | Tc productive rate (mg/ (Ld)) |
| ????12 | ????15.6±0.1??? | ????300.9±3.9 | ????19.8±0.3 * |
| ????15 | ????17.1±0.6????? | ????278.4±6.7 | ????14.3±0.2 |
| ????18 | ????21.7±0.1????? | ????303.8±1.4 * | ????13.4±0.4 |
| ????21 | ????21.9±0.8 * | ????287.7±27 | ????10.7±1.0 |
Culture condition added MJA to 100 μ M with example 1 at the 7th day, continued to be cultured to 21 days and finished fermentation.Cell density reached the highest (dry weight 19.2g/L) at the 12nd day, productive rate is 1.0g/ (Ld).The throughput of Tc sees the following form.
| Incubation time (my god) | Tc content (mg/gDW) | Tc output (me/L) | Tc productive rate (mg/ (Ld)) |
| ????12 | ????10.0±0.8 | ????199.2±18.6 | ????11.3±1.0 * |
| ????15 | ????12.1±0.7 | ????211.9±9.0 | ????9.9±0.9 |
| ????18 | ????15.8±1.7 * | ????240.0±24.9 * | ????9.8±0.7 |
| ????21 | ????15.8±0.1 | ????225.0±11.1 | ????7.7±0.6 |
Culture condition added MJA to 500 μ M with example 1 at the 7th day, continued to be cultured to 21 days and finished fermentation.Cell density reached the highest (dry weight 11.1g/L) at the 12nd day, productive rate is 0.4g/ (Ld).The throughput of Tc sees the following form.
| Incubation time (my god) | Tc content (mg/gDW) | Tc output (mg/L) | Tc productive rate (mg/ (Ld)) |
| ????12 | ????19.9±0.1 | ????214.4±11.0 | ????13.8±0.6 |
| ????15 | ????25.3±0.5 | ????280.2±10.8 * | ????15.4±0.4 * |
| ????18 | ????27.4±0.3 | ????256.7±8.6 | ????11.5±0.7 |
| ????21 | ????29.5±0.4 * | ????245.2±11.4 | ????9.3±0.5 |
Culture condition is with example 1.First group in contrast; Second group at the 7th day interpolation MJA to 100 μ M; The 3rd group was added MJA to 100 μ M simultaneously at the 7th day, and added sucrose 20g/L simultaneously, continued to be cultured to 21 days and finished fermentation, and cell density reached the highest (dry weight 23.6g/L) at the 18th day, and productive rate is 0.89g/ (Ld).The Tc throughput of each group sees the following form.
First group (contrast):
Second group (inducing):
| Incubation time (my god) | Tc content (mg/gDW) | Tc output (mg/L) | Tc productive rate (mg/ (Ld)) |
| ????12 | ????12.0±0.8 | ????201.1±17.0 * | ????10.6±0.8 * |
| ????15 | ????13.4±0.8 * | ????185.4±14.9 | ????7.4±0.6 |
| ????18 | ????12.2±1.4 | ????149.7±14.3 | ????4.2±0.3 |
| ????21 | ????11.7±0.3 | ????131.4±5.7 | ????2.8±0.2 |
| Incubation time (my god) | Tc content (mg/gDW) | Tc output (mg/L) | Tc productive rate (mg/ (Ld)) |
| ????12 | ????23.0±0.8 | ????374.8±13.9 * | ????25.1±1.1 * |
| ????15 | ????25.9±1.3 * | ????348.7±10.6 | ????18.3±0.8 |
| ????18 | ????25.0±1.6 | ????281.2±19.4 | ????11.5±1.4 |
| ????21 | ????25.1±0.2 | ????278.1±1.4 | ????9.7±0.2 |
| Incubation time (my god) | Tc content (mg/gDW) | Tc output (mg/L) | Tc productive rate (mg/ (Ld)) |
| ????12 | ????25.6±4.0 * | ????423.3±28.0 | ????29.1±1.5 |
| ????15 | ????23.7±1.3 | ????526.4±9.6 | ????30.2±0.6 * |
| ????18 | ????21.8±0.5 | ????513.6±13.0 | ????24.4±0.9 |
| ????21 | ????25.3±1.3 | ????538.9±19.5 * | ????22.1±1.3 |
Claims (5)
1. a method of utilizing taxus chinensis cell to produce taxanes comprises separating from the callus of plant children stem explant induction obtaining taxus chinensis cell, it is characterized in that also comprising the steps:
(1) method that taxus chinensis cell is moved into employing routine in the liquid nutrient medium is carried out succeeding transfer culture;
(2) place the bottle that shakes that contains substratum secretly to cultivate the Chinese Ramulus et folium taxi cuspidatae wet cell that is obtained then, culture cycle is 12-25 days, adopts conventional method to collect TAXUYUNNANINE C from the culture that is obtained;
At the quick growth phase of cell in shake-flask culture early stage, adding inductor dihydro methyl jasmonate or the methyl jasmonate ultimate density of inductor to the substratum is 1-1000 μ M.
2. the method for claim 1 is characterized in that, mends sugar when adding inductor, and additional amount is 10-30g/L; Said sugar is a kind of in sucrose, glucose, fructose or the lactose.
3. the method for claim 1, it is characterized in that, the Murashige-Skoog substratum is all adopted in bottle dark cultivation of shaking of the cultivation of taxus chinensis cell and wet cell, and interpolation 0.1-1.0mg/L 6-benzyladenine, 0.05-0.5mg/L2,4-dichlorphenoxyacetic acid, 0.05-0.5mg/L naphthylacetic acid, 20-500mg/L xitix and 10-50g/L sucrose.
4. method as claimed in claim 2 is characterized in that, said sugar is sucrose.
5. the method for claim 1 is characterized in that, Chinese Ramulus et folium taxi cuspidatae wet cell shakes when secretly cultivating in the bottle, and inoculum size is the 20-200g/L weight in wet base, and temperature is 20-30 ℃.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008151526A1 (en) * | 2007-06-08 | 2008-12-18 | Dalian Institute Of Chemical Physics, Chinese Academy Of Sciences | A method of inducing taxane production |
| CN101319199B (en) * | 2007-06-08 | 2010-10-27 | 中国科学院大连化学物理研究所 | Method for cell abduction generation of taxone with Chinese yew |
| CN105368888A (en) * | 2015-11-27 | 2016-03-02 | 天津艾赛博生物技术有限公司 | Method of producing taxane through large-scale culture by Taxus cell line three-line technology |
-
2000
- 2000-09-21 CN CN 00125305 patent/CN1118578C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008151526A1 (en) * | 2007-06-08 | 2008-12-18 | Dalian Institute Of Chemical Physics, Chinese Academy Of Sciences | A method of inducing taxane production |
| CN101319199B (en) * | 2007-06-08 | 2010-10-27 | 中国科学院大连化学物理研究所 | Method for cell abduction generation of taxone with Chinese yew |
| CN105368888A (en) * | 2015-11-27 | 2016-03-02 | 天津艾赛博生物技术有限公司 | Method of producing taxane through large-scale culture by Taxus cell line three-line technology |
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