CN1304561C - Process for raising producing rate of taxad alcohol of Taxus chinensis endogeny fungus fermenting material - Google Patents
Process for raising producing rate of taxad alcohol of Taxus chinensis endogeny fungus fermenting material Download PDFInfo
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Abstract
The present invention relates to a method for enhancing taxol yield in yew endophytic fungus fermentation objects. The method utilizes 10-deacetyl baccatin III and baccatin III which can generate taxol endophytic fungi and taxol synthesis precursors for respective and single culture; then, culture objects are cultured in a mixed mode, and a culture medium is appropriately added; the taxol yield in fermentation objects is largely enhanced. The method has the advantages that taxol contents in the fermentation objects are largely enhanced by the metabolic pathway complementation of two kinds of fungi, and are largely higher than the yield level of single culture of producing taxol endophytic fungi. The industrialized fermentation culture for generating taxol by endophytic fungi is possible. The method is suitable for the mixed culture of endophytic fungi of generating taxol endophytic fungi and taxol synthesis precursors, and taxol contents in the culture objects are enhanced. The method is also suitable for the mixed culture of endophytic fungi for generating endophytic fungi of natural useful components and synthesis precursors of natural useful components, and the contents of natural useful components in culture objects are enhanced.
Description
Technical field
Present method is to utilize the endogenetic fungus culture of taxol-producing endophytic fungi culture and paclitaxel produced synthetic precursor to carry out mixed culture, increases substantially a kind of method of content of taxol in its fermented product, belongs to biological technical field.
Background technology
Extract the bark from wani et al. from yewtree (Taxus brevifolia Nutt.) and isolate taxol, and find that it has had since the antitumous effect, the scientific research personnel finds its unique anticancer mechanism again successively and can treat multiple cancer.Therefore, people begin in large quantities to extract taxol to satisfy the demand of medical market from the bark of Ramulus et folium taxi cuspidatae.Because Chinese yew worldwide belongs to rare plant,, jeopardize the eubiosis from bark so taxol is inevitable seriously to destroy natural resources by extracting.In order to solve medicine source short supply state, Chinese scholars has been done big quantity research and is comprised and cultivate new variety, chemical synthetic, tissue culture, cell cultures and microbial fermentation for obtaining taxol by all kinds of means.
Stroble and Stierle in reported first in 1993 from the Pacific yew tree, isolate a kind of endogenetic fungus that can be paclitaxel produced, its content of taxol is 24～50ng/L, for people have showed the tempting new way that may produce taxol.Afterwards, people have isolated many endogenetic fungus that can the fermentative production taxol from various taxaceae plants, the content of taxol of report is about 14.2-406.95 μ g/L, wherein, Ma Tianyou, the taxol yield of Dong Zhaolin report is 14.2ug/L, (" Northwest University's journal (natural science edition) ", 1999,29 (1): 47～49); Strobel G A, Hess W M, Ford E etc. utilize the productive rate of little spore dish stey fermentation to be 60-79ug/L (J Ind Microb, 1996,17:417 ~ 423); JY Li, it is (Joumal of induction Microbiology ﹠amp about 85.1 μ g/L that RS Sidhu etc. utilizes the endogenetic fungus fermentation yield; Biotechnology.1998,20,259-264); Wang Jianfeng, Lv Huaying, Huang Yaojian etc. the taxol yield that utilizes the fermentation of knurl seat spore to obtain is 185.4ug/L, (" Xiamen University's journal (natural science edition) ", 1999,38 (4): 485～487); It is 406.950 μ g/L (" fungus system ", 2002,21 (2): 246-251) that Liu Xiaolan, Zhou Dongpo, Sun Jianqiu etc. utilize tree-shaped more piece spore fermentation yield.These fermentation yield levels are very low, also have certain gap apart from suitability for industrialized production.In general, the yield level of taxol will reach and just has industrial production to be worth about 1mg/L in the fermented product.
Summary of the invention
The purpose of present method is to provide a kind of endogenetic fungus culture by taxol-producing endophytic fungi culture and paclitaxel produced synthetic precursor to carry out the novel method that mixed culture is produced taxol, makes that content of taxol increases substantially in its fermented product.
Method of the present invention is: go the culture of endogenetic fungus (the Ozonium sp.Lk.ex Fr.) XT1 of acetyl baccatin III and baccatin III to carry out mixed culture taxol-producing endophytic fungi (Ectostromasp.Fr.) XT5 of cauline leaf pyrenomycetes genus and the paclitaxel produced synthetic precursor 10-of synnema Pseudomonas, increase substantially the productive rate of taxol in the fermented product.
Method mainly adopts following steps:
1, medium preparation
(1) PDA substratum.
(2) fermention medium:
Glucose 2-10g/L sucrose 10-20g/L peptone 0.5-2.5g/L
Sodium-acetate 0.5-3g/L MgSO
42-5mg/L Ca (NO
3)
25-100mg/L
CuSO
40.5-300mg/L ZnSO
40.5-200mg/L MnCl
21-25mg/L
FeCl
30.5-10mg/L KH
2PO
4The 10-350mg/L vitamins B
10.5-10mg/L vitamins B
60.5-30mg/L phenylalanine 0.5-5mg/L Sodium Benzoate 50-1000mg/L Sodium o-acetyloxybenzoate 0.01-5mg/L methyl alcohol 5-20ml/L oleic acid 1-10ml/L.pH 6.8。
2, thalline is cultivated in advance.Picking taxol-producing endophytic fungi and paclitaxel produced synthetic precursor 10-go the mycelium inoculation of acetyl baccatin III and baccatin III endogenetic fungus in the fermentation flask that contains 50ml PDA substratum respectively, at 28 ℃, 200 rpms of shaking table dark culturing 2d form seed fermentation liquid.
3, thalli growth is cultivated.Respectively the seed fermentation liquid of above-mentioned two kinds of fungies is inoculated in and contains the shaking in the bottle of 500ml fermention medium, on the constant temperature shaking table in 28 ℃, 200rpm dark culturing 3d.The enlarged culturing scale can be carried out diauxy and be cultivated.
4, mixed culture is produced taxol.The taxol-producing endophytic fungi culture that growth is good and the endogenetic fungus culture of paclitaxel produced synthetic precursor by 1: 0.5～4.0 volume parts than mixing, add the 0.5-1.0 fermention medium doubly of cumulative volume, on the constant temperature shaking table in 28 ℃, 200rpm dark culturing 6d.
5, the extraction of taxol: with the centrifugal 15min of fermented product 4000rpm.Supernatant liquor and low temperature freeze molten thalline and add isopyknic ethyl acetate ultrasonic extraction 1h, repeat to extract 2 times.Merge all ethyl acetate layers, in 50 ℃ of rotation evaporates to dryness, methanol wash after (v/v) extraction in 1: 1 of adding methylene dichloride and water, with the methylene dichloride evaporate to dryness, is accurately measured the 1ml dissolve with ethanol and is obtained the taxol sample.
6, content detection: go up performance liquid chromatographic column behind the taxol sample filtering, do qualitative and detection by quantitative in 228nm.Qualitative with retention time, carry out quantitative analysis with external standard method.Standard substance are Sigma company product, and high-efficient liquid phase chromatogram condition is: mobile phase methanol-water (65: 35), reverse C18 post, column length 15cm, 25 ℃ of column temperatures, flow velocity 0.8ml/min.
Taxol-producing endophytic fungi described in present method comprises:
XT5 (Ectostroma sp.Fr.), the strain classification status is accredited as the cauline leaf pyrenomycetes and belongs to (645-649 in 1979 of fungi identification handbook Wei Jing super posthumous work Shanghai Shanghai science tech publishing house)
TAX-47 (stem point Pseudomonas Phoma Sacc) (reference: Chen Yi's heavily fortified point, Zhang Shuo, Wang Yan etc. the screening of paclitaxel produced fungi in taxusyunnanensis (Taxus yunnanensis) endogenetic fungus. biotechnology .2003,13 (2) .-10-11);
TAX-49 (the cocainine pyrenomycetes belongs to Phacodium Pers.Ex Wallr.) (reference: Chen Yi's heavily fortified point, Zhang Shuo, Wang Yan etc. the screening of paclitaxel produced fungi in taxusyunnanensis (Taxus yunnanensis) endogenetic fungus. biotechnology .2003,13 (2) .-10-11);
Tree-shaped more piece spore Nodulisporium sylviforme (reference: Zhou Dongpo, flat literary composition is auspicious, Sun Jianqiu etc. and taxol produces the isolating research of bacterium. JOURNAL OF MICROBIOLOGY, 2001,21 (1): 18 ~ 20);
Knurl seat spore Tubercularia sp. (reference: Wang Jianfeng, Lv Huaying, Huang Yaojian etc. the new taxol of a strain produces bacterium and anti-tumor activity thereof. Xiamen University's journal (natural science edition), 1999,38 (4): 485 ~ 487),
Little spore dish stey Pestalotiopsis mictospora (reference: Strobel G, YangX S, Sars J et al.Taxol from Pestalotiopsis microspora, an endophyticfungus of Taxus wallachiana.Microbiology, 1996,142:435 ~ 440) etc.
Described paclitaxel produced synthetic precursor 10-goes acetyl baccatin III and baccatin III endogenetic fungus to comprise:
XT1 (Ozonium sp.Lk.ex Fr.), the strain classification status is accredited as synnema Pseudomonas (645-649 in 1979 of fungi identification handbook Wei Jing super posthumous work Shanghai Shanghai science tech publishing house);
TAX-25 (the cauline leaf pyrenomycetes belongs to Ectostroma Fr.) (reference: Chen Yi's heavily fortified point, Zhang Shuo, Wang Yan etc. the screening of paclitaxel produced fungi in taxusyunnanensis (Taxus yunnanensis) endogenetic fungus. biotechnology .2003,13 (2) .-10-11) etc.
Present method is to utilize this two kinds of similar fungi pathways metabolisms complementation, the endogenetic fungus that makes on the one hand high paclitaxel produced synthetic precursor makes full use of the bio-transformation ability taxol biosynthesis of taxol-producing endophytic fungi bacterial strain for the bio-transformation of taxol-producing endophytic fungi provides a large amount of synthetic precursors (10-removes acetyl baccatin III and baccatin III).Taxol-producing endophytic fungi provides the taxol synthetic enzyme and the signal induction factor for the endogenetic fungus of the paclitaxel produced synthetic precursor of height on the other hand, make a large amount of 10-of taxol-producing endophytic fungi bacterial strain synthetic go acetyl baccatin III and baccatin III to be converted into taxol, the productive rate of taxol improves greatly in the fermented product thereby make.Grow latter stage at endogenetic fungus, promote the increase of cell permeability and the self-dissolving that the part thalline takes place, also created condition for the productive rate that increases substantially taxol.
Utilize present method can significantly improve the content of taxol, content of taxol in the fermented product is improved about 3.6-4.0 times, reach between the 731.3022 μ g/L-1094.0274 μ g/L, the yield level that is much higher than the taxol-producing endophytic fungi single culture makes and utilizes endogenetic fungus to carry out industrial fermentation cultivate to produce taxol and become possibility.
Present method not only is applicable to the endogenetic fungus mixed culture of taxol-producing endophytic fungi and paclitaxel produced synthetic precursor, improves content of taxol in the culture.Also be suitable for producing the endogenetic fungus and the endogenetic fungus mixed culture that produces the synthetic precursor of natural useful component of natural useful component, improve the content of natural useful component in the culture.
Embodiment
Embodiment 1:
This example is selected for use is that the culture of isolated two strain endogenetic fungus XT1 from southerm yew (high paclitaxel produced synthetic precursor 10-removes acetyl baccatin III and baccatin III, but paclitaxel produced hardly) and XT5 (paclitaxel produced) carries out mixed culture production taxol.
Two endophyte strain kinds that this example relates to identify that (645-649 in 1979 of fungi identification handbook Wei Jing super posthumous work Shanghai Shanghai science tech publishing house) is as following table:
The morphological specificity of endogenetic fungus, product and classification position
| The bacterial classification code name | Host plant | Product | Morphological specificity | Classification position |
| XT1 | Southerm yew Taxus chinnsis Var.mairei | 10-removes acetyl baccatin III and baccatin III | Has only infertile mycelia, finding no property and asexual spore, mycelium is flocculence, do not find sclerotium, mycelia is thick, be woven into nettedly, thalline is white in color in early days, and the later stage is light beige | Synnema Pseudomonas Ozonium Lk.ex Fr. |
| XT5 | Southerm yew Taxus chinnsis var.mairei | Taxol | Has only infertile mycelia, finding no property and asexual spore, later stage can produce the dark-coloured sclerotium of circular fine granularity, the outer differentiation of sclerotium is not obvious, and the center is colourless. | The cauline leaf pyrenomycetes belongs to Ectostroma Fr. |
Above-mentioned XT5 can adopt existing conventional method to separate acquisition with XT1, below exemplifies a kind of separation method:
The bark of southerm yew (Taxus chinansis var maurei) is cut into the segment of about 1.5cm, respectively through alcohol surface sterilization 5～15min of 75%, 0.1% mercuric chloride solution is handled 15min, use aseptic water washing then, be inoculated on the PDA solid medium plate that contains Streptomycin sulphate, be positioned in 25 ℃ of thermostat containers and cultivate, after waiting to grow bacterium colony, choose by the aseptic technique program, at purifying on the PDA slant medium more than 3 times.The fungal bacterial strain that obtains after the separation and purification carries out the shaking table fluid enlargement culture then, extract taxol and various precursor in the fermented product, utilize high-performance liquid chromatogram determination, retention time according to taxol and the synthetic precursor standard substance of taxol is qualitative, quantitative with external standard method, the endogenetic fungus XT5 that filters out can produce taxol, removes acetyl baccatin III and baccatin III and XT1 can produce the synthetic precursor of taxol: 10-, and the content height, but produce taxol hardly.
Below be detailed process with XT5 and XT1 mixed culture extraction taxol:
1, medium preparation:
The preparation of PDA substratum: every liter contains 200g potato poach supernatant liquor and 20g glucose, the pH nature.
Fermention medium is formed (unit of mark be mg/L):
Glucose 5g/L sucrose 15g/L peptone 1g/L
Sodium-acetate 1g/L MgSO
42.5mg/L Ca (NO
3)
210mg/L
CuSO
410mg/L ZnSO
45mg/L MnCl
22.5mg/L
FeCl
32mg/L KH
2PO
4The 100mg/L vitamins B
15mg/L
Vitamins B
65mg/L phenylalanine 2mg/L Sodium Benzoate 400mg/L
Sodium o-acetyloxybenzoate 1mg/L methyl alcohol 10ml/L oleic acid 5ml/L
PH is transferred to 6.8, adds during the Sodium o-acetyloxybenzoate mixed culture again.
2, thalline is cultivated in advance.Picking XT1 and XT5 mycelium inoculation be in the fermentation flask that contains 50ml PDA substratum respectively, and on the constant temperature shaking table 28 ℃, 200rpm dark culturing 2d forms ferment-seeded.
3, thalli growth is cultivated.Respectively the seed fermentation liquid of XT1 and XT5 is inoculated in and contains the 500ml fermentation with the shaking in the bottle bottle of substratum, on the constant temperature shaking table 28 ℃, 200rpm dark culturing 3d.Be the enlarged culturing scale, can carry out diauxy and cultivate.
4, mixed culture is produced taxol.Taxol-producing endophytic fungi that growth is good and high paclitaxel produced synthetic precursor endogenetic fungus be single culture respectively, mix by 1: 0.5～4.0 volume parts ratio then, add the 0.5-1.0 fermention medium doubly of cumulative volume, on the constant temperature shaking table 28 ℃, 200rpm dark culturing 6d.
5, taxol extracts: with the centrifugal 15min of fermented product 4000rpm.Behind 60 ℃ of rotary evaporation to 1/10 volumes of supernatant liquor, add isopyknic ethyl acetate and under ultrasonic wave, extract 1h; Handle the back isopyknic ethyl acetate of adding with tissue mincer after thalline low temperature freezes molten 2 times and extract 1h under ultrasonic wave, collect ethyl acetate layer, bacterial sediment and supernatant liquor add isopyknic ethyl acetate ultrasonic extraction 2 times again.Merge all ethyl acetate layers, to doing, add an amount of dissolve with methanol washing solids in 50 ℃ of rotary evaporations, after 1: 1 (v/v) extraction that adds methylene dichloride and water again, dichloromethane layer is extremely done in 45 ℃ of rotary evaporations, accurately measured the 1ml dissolve with ethanol and obtain the taxol sample.
6, content detection: go up performance liquid chromatographic column behind the taxol sample filtering, do qualitative and detection by quantitative in 228nm.Qualitative with retention time, carry out quantitative analysis with external standard method.Standard substance are Sigma company product, and high-efficient liquid phase chromatogram condition is: mobile phase methanol-water (65: 35), reverse C18 post, column length 15cm, 25 ℃ of column temperatures, flow velocity 0.8ml/min.
The contrast of above-mentioned mixed culture and two bacterial strain single culture is compared, the mixed culture fermented product, the XT5 single culture, content of taxol is respectively in the fermented product of XT1 single culture: 731.3022 μ g/L, 191.3381 μ g/L, 0 μ g/L, the content of taxol that can extract in the fermented product of mixed culture is apparently higher than the fermented product of bacterial strain single culture.
Embodiment 2:
Change sucrose in the fermention medium in the example 1 into 10g/L, phenylalanine changes 1mg/L into, and Sodium o-acetyloxybenzoate changes 0.5mg/L into, and oleic acid changes 10ml/L into, other conditions are constant, carry out XT5 and XT1 mixed culture, XT5, XT1 single culture, high performance liquid chromatography detects, content of taxol is respectively in the fermented product: 1094.0274 μ g/L, 276.7554 μ g/L, 0 μ g/L.
Table: the μ of content of taxol unit g/L in the culture
| Example number | XT1 or cocainine pyrenomycetes belong to single culture | XT5 or cauline leaf pyrenomycetes belong to single culture | Mixed culture | Mixed culture improves multiple |
| 1 | 0 | 191.3381 | 731.3022 | 3.8220 |
| 2 | 0 | 276.554 | 1094.0274 | 3.9530 |
Claims (1)
1, a kind of method that improves taxol yield in the Ramulus et folium taxi cuspidatae endogenetic fungus fermented product, it is characterized in that: go the culture of endogenetic fungus (Ozonium sp.Lk.exFr.) XT1 of acetyl baccatin III and baccatin III to carry out mixed culture taxol-producing endophytic fungi (Ectostroma sp.Fr.) XT5 of cauline leaf pyrenomycetes genus and the paclitaxel produced synthetic precursor 10-of synnema Pseudomonas, improve the productive rate of taxol in the fermented product, step is as follows:
(1) medium preparation
1. PDA substratum:
2. fermention medium:
Glucose 2-10g/L sucrose 10-20g/L peptone 0.5-2.5g/L
Sodium-acetate 0.5-3g/L MgSO
42-5mg/L Ca (NO
3)
25-100mg/L
CuSO
40.5-300mg/L ZnSO
40.5-200mg/L MnCl
21-25mg/L
FeCl
30.5-10mg/L KH
2PO
4The 10-350mg/L vitamins B
10.5-10mg/L
Vitamins B
60.5-30mg/L phenylalanine 0.5-5mg/L Sodium Benzoate 50-1000mg/L
Sodium o-acetyloxybenzoate 0.01-5mg/L methyl alcohol 5-20ml/L oleic acid 1-10ml/L, pH 6.8;
(2) thalline is cultivated in advance; Respectively the paclitaxel produced synthetic precursor 10-of picking synnema Pseudomonas remove the taxol-producing endophytic fungi XT5 that the endogenetic fungus XT1 of acetyl baccatin III and baccatin III and cauline leaf pyrenomycetes belong to mycelium inoculation in the fermentation flask that contains 50ml PDA substratum, cultivate and form seed fermentation liquid;
(3) thalli growth is cultivated: seed fermentation liquid that respectively will above-mentioned two kinds of fungies is inoculated in and contains the shaking in the bottle of 500ml fermention medium, and cultivation obtains the fungi bacterium liquid of mycetome and nutrient solution;
(4) with above-mentioned two kinds of fermented products, promptly fungi bacterium liquid carries out mixed culture and produces taxol: will grow good XT5 bacterium liquid and XT1 bacterium liquid than mixing, are added 0.5-1.0 times fermention medium cultivation of cumulative volume by 1: 0.5～4.0 volume parts;
(5) taxol extracts: with the centrifugal 15min of above-mentioned fermented product 4000rpm, supernatant liquor and low temperature freeze molten thalline and add isopyknic ethyl acetate ultrasonic extraction 1h, repeat to extract 2 times; Merge all ethyl acetate layers, in 50 ℃ of rotation evaporates to dryness, methanol wash after adding methylene dichloride and water 1: the 1v/v extraction, with the methylene dichloride evaporate to dryness, is accurately measured the 1ml dissolve with ethanol and is obtained the taxol sample;
(6) content detection: go up performance liquid chromatographic column behind the taxol sample filtering, do qualitative and detection by quantitative in 228nm.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932024B (en) * | 2006-09-15 | 2010-09-29 | 清华大学 | Taxol biosynthesizing process and special culture medium |
| CN101386874B (en) * | 2008-10-28 | 2011-05-11 | 南开大学 | Two-liquid phase fermentation method for improving paclitaxel yield from fungi |
| CN103865800B (en) * | 2012-12-11 | 2016-04-27 | 河南科技学院 | The Ramulus et folium taxi cuspidatae endogenetic fungus of baccatin III is produced in one strain |
| CN104726345B (en) * | 2015-02-07 | 2020-12-04 | 河南科技学院 | High-yield baccatin III combined strain and method for producing baccatin III |
| CN104975054A (en) * | 2015-06-25 | 2015-10-14 | 河南科技学院 | Method for improving yield of 10-deacetylbaccatin III produced by fungi |
| CN106818256A (en) * | 2016-12-12 | 2017-06-13 | 浙江大学宁波理工学院 | Improve the regulation and control method of yew alkanes material in leaves of Taxus mairei |
| CN110302215A (en) * | 2019-05-15 | 2019-10-08 | 中国人民解放军陆军军医大学第一附属医院 | Taxus x media endogenetic fungus crude extract and application |
| CN113736835A (en) * | 2021-09-10 | 2021-12-03 | 陈开云 | Industrial preparation process for producing taxol by fermenting taxus chinensis endophytic fungi mutant |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1158356A (en) * | 1996-11-27 | 1997-09-03 | 天津大学 | Method for improving content and release of yew alcohol in Taxus chinensis cell |
| 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 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1158356A (en) * | 1996-11-27 | 1997-09-03 | 天津大学 | Method for improving content and release of yew alcohol in Taxus chinensis cell |
| 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 |
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