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Industrial preparation process for producing paclitaxel by fermenting taxus endophytic fungus mutant

A technology of endophytic fungi and preparation technology, which is applied in the field of bio-metabolism synthesis and purification, can solve the problems of no industrial production of paclitaxel, no prospect of industrial application, low yield of paclitaxel compounds, etc., and achieve large production capacity, low product cost, The effect of optimizing energy consumption

Pending Publication Date: 2021-12-03
陈开云 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the yield of paclitaxel is only 16.59 mg / L, and the production cost is much higher than that of paclitaxel extracted from Taxus chinensis. Therefore, the patented technology still does not meet the conditions for industrial production of paclitaxel
[0009] The above two patented technologies utilize the taxus endophytic fungi to ferment paclitaxel. Although the paclitaxel is produced by fermenting the taxus endophytic fungi, the reaction conditions are mild, the pollution is small, and the operation is relatively simple. However, the yield of paclitaxel compounds is very low, so , still does not have the prospect of industrial application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0102]The preparation and industrial fermentation method of bacterial classification are as follows:

[0103] i. The formula of liquid shake flask culture medium is PDA, pH 7, initial pH value is 5.0, a total of 250ml is prepared, divided into 250mL triangular flasks, each bottle is 30ml, sterilized at 121°C for 30 minutes, then cooled to room temperature for use;

[0104] ii. Pick and transfer the cultured strains on the slant of the test tube into a 250ml Erlenmeyer flask equipped with a shaker culture medium, and place it in a shaking table at 28°C and 120 rpm for 24 hours;

[0105] iii. Prepare the seed culture medium according to the formula, the formula of the described seed culture medium is 40g of sucrose, 10g of peptone, 10g of yeast extract powder, 15g of agar, 1000mL of distilled water, pH 7; the seed culture medium is pumped into No. 1-8 500L- Grade seed culture tanks, sterilized by steam sterilization in real tanks, with a sterilization temperature of ≥121°C and a...

Embodiment 1

[0162] The isolation, mutagenesis and preservation method steps of Trichoderma HQ-24 bacterial strain are as follows:

[0163] a. Using the conventional tissue separation method, cut the fresh Taxus chinensis fruit in Yunnan area into 0.5-1cm 2 small pieces. Soak the fruit in 75% alcohol for about 5 minutes, rinse it with sterile water for 3 to 4 times, and then soak it with 2.5% sodium hypochlorite for 3 minutes; plant the contact culture medium on the cutting part, and use the tissue imprinting method as a control to test whether the surface is disinfected. thorough;

[0164] b. Culture the culture medium in a constant temperature incubator at 28°C. Observe regularly, transfer the newly grown hyphae to the new solid medium in turn with an inoculation needle, purify each strain 2 to 3 times, and then transfer to the slant of the test tube to store two of each;

[0165] c. Preparation of bacterial suspension: Take a few slants of dominant strains cultured for one week, add ...

Embodiment 2

[0201] Strain preparation and industrial fermentation method:

[0202] i. The formula of liquid shake flask culture medium is PDA, pH 7. The initial pH value is 5.0. A total of 250ml was prepared, divided into 250mL Erlenmeyer flasks, 30ml per bottle, sterilized at 121°C for 30 minutes, then cooled to room temperature for use;

[0203] ii. Pick and transfer the cultured strains on the slant of the test tube into a 250ml Erlenmeyer flask equipped with a shaker culture medium, and place it in a shaking table at 28°C and 120 rpm for 24 hours;

[0204] iii. Prepare the seed culture medium according to the formula standard, pump the seed culture medium into the No. 2 500L first-level seed culture tank, and sterilize it by steam sterilization in real tank, the sterilization temperature is ≥121℃, and the sterilization time is ≥30min;

[0205] iv. After the sterilization of the first-level seed culture tank is completed, the temperature of the culture medium in the tank is lowered t...

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Abstract

The invention discloses an industrial preparation method for producing paclitaxel by fermentation of taxus endophytic fungi. The method comprises the following steps that a trichoderma HQ-24 strain is separated from taxus fruits; and the taxus endophytic fungi are fermented to produce a paclitaxel compound under the synergistic effect of mutagenesis, test tube slant culture, seed culture, fermentation culture, membrane filtration, membrane concentration, extraction separation, macroporous resin purification, recrystallization and other technologies, and a supercritical CO2 fluid drying technology and a full-automatic control technology are combined. The non-chemical synthesis of the paclitaxel compound and the biosynthesis of non-taxus extraction are realized; and the method for producing the paclitaxel compound by utilizing the technology is mild in reaction condition, relatively simple to operate, low in product cost, high in yield, high in purity, low in residual solvent, low in energy consumption, large in productivity, small in environmental pollution and suitable for industrial large-scale production, and has good economic benefits and application prospects.

Description

technical field [0001] The invention belongs to the technical field of biological metabolism synthesis and purification, and specifically relates to an industrial preparation process for paclitaxel produced by fermenting taxus endophytic fungus mutants. Background technique [0002] Paclitaxel (registered name Taxol) is a diterpene derivative, a natural product with a taxane ring structure with a unique anti-leukemia and anti-cancer mechanism, and was first isolated from Taxus. Paclitaxel is a white crystalline powder, insoluble in water, easily soluble in organic solvents such as methanol, ethanol, acetone, ethyl acetate, ether, etc., a tricyclic diterpenoid compound composed of 113 atoms, molecular formula C47H51NO14, relative molecular weight 853.92, melting point 213 -216°C. [0003] Paclitaxel is an important anticancer drug for clinical application. Taxanes are metabolic precursors or side metabolites of paclitaxel. Some taxanes have special physiological activities. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/02C12N13/00C12N15/01C12M1/38C12M1/36C12M1/04C12M1/12C12M1/00C07D305/14C12R1/885
CPCC12P17/02C12N13/00C12N15/01C12M23/58C12M29/26C12M41/12C12M41/48C12M41/44C12M37/00C12M39/00C07D305/14
Inventor 陈开云柏凤静
Owner 陈开云