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Aspergillus candidus for producing taxol and method for preparing taxol from same

A technology of Aspergillus leucoides and paclitaxel, which is applied in the field of preparing anticancer drug paclitaxel, and can solve the problems of low yield of paclitaxel and the like

Inactive Publication Date: 2009-11-25
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But, the paclitaxel production rate of the paclitaxel-producing fungi found so far is all relatively low, and the taxol production rate of Taxomyces andreanae (Taxomyces andreanae) found for the first time is only 24~50ng / L (Stierle et al., 1993, Science, 260:214- 216)

Method used

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  • Aspergillus candidus for producing taxol and method for preparing taxol from same
  • Aspergillus candidus for producing taxol and method for preparing taxol from same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Collect the bark tissues of Taxus chinensis planted in the campus nursery of Huazhong University of Science and Technology, cut them into small pieces (~0.5×0.5×0.5cm) with a sterile knife, disinfect the surface with 70% (v / v) alcohol for 3 minutes, and use sterilized ddH 2 Rinse three times with O, then peel off the inner epidermis with a sterile knife and evenly place it on a PDA medium plate containing 50 μg / mL ampicillin, and culture it statically in an incubator at 25°C to 28°C. , the fungi with different morphology were isolated on a new PDA medium plate by the separation and purification method of the top of the hyphae. The isolated fungi were cultured and subcultured at 25°C to 28°C, and their purity was tested until a single clone was obtained. Finally, the single clone of each fungus was inoculated to a new PDA slant for preservation by the mycelium top separation method again, and the spores of the corresponding fungi were collected to prepare a spore suspen...

Embodiment 2

[0025] Use an inoculation needle to pick a little of Aspergillus leucobacter HUST-RBB3 preserved on the PDA slant, inoculate it into 300ml potato glucose liquid medium (1L Erlenmeyer flask), and cultivate it for 10 days at 25°C and 180rpm. Centrifuge the fermentation broth at 4°C and 12,000rpm for 10 minutes, collect the mycelia and fermentation broth respectively, and prepare 2ml of crude paclitaxel dissolved in 100% methanol according to steps (3) to (5) in the above optimization scheme. liquid. After purification according to the conventional paclitaxel purification method (Yu Longjiang, et al. 2002, Forestry Chemistry and Industry, 22: 45-48), a paclitaxel solution dissolved in 1 ml of 100% methanol was obtained. Take 10 μ l for HPLC analysis, the instrument is Agilent 1200 HPLCSystem, the chromatographic column is a C18 reverse column (5 × 300mm, Waters), and the mobile phase is methanol: H 2 O (68:32, v / v), the flow rate is 1.0 ml / min, the ultraviolet wavelength is set ...

Embodiment 3

[0027]Take 10 μl of paclitaxel isolated and purified after fermentation of the Aspergillus leucobacter HUST-RBB3 strain obtained in Example 2 for mass spectrometry analysis (completed by the Analysis and Testing Center of Huazhong University of Science and Technology). The mass spectrometer is Agilent 1100 LC / MSD Trap, using electrospray mass spectrometry (electrospray massspectrometry) technology, spray voltage (a spray voltage): 2.2kV; column type: C18 separation column (5 × 300mm, Waters); injection volume: 10μl ; Detection wavelength: 227nm; Mobile phase: methanol: H 2 O (68:32, v / v); flow rate: 1ml / min, the standard paclitaxel was used as the control. The results showed that the paclitaxel produced by the Aspergillus leucobacter HUST-RBB3 strain had the same molecular weight as the paclitaxel standard isolated from the natural Taxus chinensis plant.

[0028] Get 500 μl of paclitaxel isolated and purified after the fermentation of the obtained Aspergillus leucobacter HUST...

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Abstract

The invention relates to Aspergillus candidus which is separated from Taxus x media and is used for producing taxol. Under the condition of shaking culture at the temperature of 25 DEG C and with the rotating speed of 180 rpm, a wild strain Aspergillus candidus HUST-RBB3 is fermented for 10 days in 300 mL of potato glucose liquid culture medium to obtain the taxol with a concentration of about 120 mu g / L, and is a wild strain with higher taxol yield under unoptimized culture conditions which has been reported at present. Under preliminarily optimized fermentation conditions, the fermentation of the Aspergillus candidus HUST-RBB3 can produce the taxol with a concentration of about 497.3 mu g / L. The production of the taxol through the fermentation of the strain has the advantages of short production period, easy strain breeding and preservation and the like. The invention provides a novel strain and corresponding fermentation production technology for producing the taxol by utilizing microbial fermentation.

Description

technical field [0001] The invention belongs to the field of microbial biotechnology and biopharmaceuticals, and in particular relates to a taxol-producing Aspergillus candidus and a paclitaxel-producing Aspergillus candidus HUST-RBB3 strain, and a method for preparing the anticancer drug paclitaxel using the same. Background technique [0002] Paclitaxel isolated from the bark of Taxus is currently recognized as an important anti-tumor drug in the world, but due to the shortage of Taxus resources, the source of Taxol is in short supply. The production of paclitaxel by microbial fermentation is one of the most effective ways to solve the problem of paclitaxel drug sources. Paclitaxel produced by microbial fermentation has the advantages of easy cultivation, easy control, and low production cost. The production of paclitaxel can be greatly increased by optimizing fermentation conditions and other means. Its large-scale industrial production is relatively easy to achieve and h...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12P17/02C12R1/66
Inventor 张鹏余龙江周蓬蓬
Owner HUAZHONG UNIV OF SCI & TECH
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