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Microorganism for hydrolyzing 7-xylose group and 13-side chain of taxane

A technology of taxanes and taxanes, which is applied in the field of enzymes and taxanes, can solve the problems of unstable production capacity of yew cells, complicated preparation process of taxanes, and inability to industrialize production. The effect of large-scale industrial production, strong biological activity, and simplified preparation process

Inactive Publication Date: 2010-03-31
INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] (4) Production of paclitaxel by cell culture method: paclitaxel and its synthetic precursors are produced by yew cells cultured in vitro, but industrial production is not yet possible due to the unstable production capacity of yew cells
[0014] In the analysis of the existing literature, there are currently published reports on such studies, but the microbial strains involved can only catalyze one reaction, namely the hydrolysis of xylose at C-7 position or the hydrolysis of side chain at C-13 position
However, no bacterial strain with dual functions has been seen at present, resulting in complex preparation process of taxanes, high cost, and low yield of taxanes

Method used

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  • Microorganism for hydrolyzing 7-xylose group and 13-side chain of taxane
  • Microorganism for hydrolyzing 7-xylose group and 13-side chain of taxane
  • Microorganism for hydrolyzing 7-xylose group and 13-side chain of taxane

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Screening and Identification of Enterobacter cloacae

[0071] More than 200 strains of microorganisms derived from the soil samples below 5cm below the surface of the yew trees in Northeast China were screened for the ability to hydrolyze the xylose group of 7-xylose-10-deacetyltaxol. The results showed that the strain Enterobacter cloacae has 7- Hydrolysis capacity of the xylose group of xylose-10-desacetylpaclitaxel.

[0072] The isolated microorganisms were stored on the slant of potato dextrose agar (PDA). During the preliminary screening of microorganisms, each bacterial strain was cultivated in two Erlenmeyer flasks filled with wheat bran medium. After cultivating for 2 days, only DMF (N, N-dimethylformamide) solvent was added to one bottle as a blank control. Another bottle was added with a DMF solution of the substrate 7-xylose-10-desacetylpaclitaxel. After continuing the cultivation for 7 days, the filtered culture solution was extracted with ethyl...

Embodiment 2

[0095] Example 2: Effects of different culture media on the conversion of 7-xylose-10-deacetylpaclitaxel

[0096] Use the following 4 media as the transformation system for microorganisms:

[0097] Medium A: wheat bran 50g, K 2 HPO 4 2g, (NH 4 ) 2 HPO 3 2 g, MgSO 4 7H 2 O, 0.1g, distilled water to 1000ml;

[0098] Medium B: beef extract 10g, NaCl 5g, peptone 10g, water 1000ml;

[0099] Medium C: Glucose (or one of α-lactose, galactose, sucrose, mannitol, fructose, glycerol, inositol, sorbitol, and D-xylose) 20g, peptone 2g, yeast extract 2g, K2 HPO 4 1g, KH 2 PO 4 1g, MgSO 4 ·7H 2 O 0.2g, FeSO 4 ·7H 2 O 0.01g, NaCl 0.01g, MnSO 4 4H 2 O 0.01g;

[0100] Medium D: 200g potatoes (boil for 20min and filter), 20g glucose, 1000ml water;

[0101] Prepare 50ml of each of the above four culture media and put them into a 250ml Erlenmeyer flask, and adjust the pH value to 6.0. Under sterile conditions, add 1 ml of Enterobacter cloacae suspension cultured for 2 days ...

Embodiment 3

[0105] Example 3: Effects of different concentrations of ammonium sulfate on the hydrolysis of the xylose group of 7-xylose-10-deacetyltaxol

[0106] Preparation of wheat bran medium: wheat bran 50g, K 2 HPO 4 2g, (NH 4 ) 2 HPO 3 2 g, MgSO 4 7H 2 O, 0.1 gram, distilled water to 1000ml; Add ammonium sulfate in the wheat bran culture medium, adopt 5 (NH 4 ) 2 SO 4 Concentrations are 0, 0.4%, 1%, 2%, 3%, and the corresponding numbers are N 0 , N 1 , N 2 , N 3 , N 4 . The prepared each (NH 4 ) 2 SO 4 Concentration medium, fill 50ml wheat bran medium in every 250ml Erlenmeyer flask. Operation method is the same as embodiment 2. The content of the conversion product was detected by HPLC. The residue obtained after concentration was dissolved in methanol and the volume was adjusted to 10 ml, and filtered as the HPLC test solution. Three parallels were performed for each concentration gradient.

[0107] 1) Preparation of standard solution: Accurately weigh 6.1 m...

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Abstract

The invention relates to enterobacter cloacae which are found in soil and have the collection number of CGMCC No.2487 or resting cell with equivalent function, variant and mutant as well as enzyme produced thereby. The invention also provides a preparation method of taxane, namely comprising the following steps: the enterobacter cloacae are used for hydrolyzing 7-xylose group in 7-xylose taxane molecule in wheat bran culture medium, and 13-side chain in taxane molecule with 13-side chain can be hydrolyzed in peptone beef extract culture medium; and the produced taxane can be used for preparing antitumor drugs, such as intermediates of taxane drugs, docetaxel drugs or taxane drugs.

Description

Technical field: [0001] The present invention relates to a kind of preparation method of Enterobacter cloacae (Enterobactercloacae) or its functionally equivalent quiescent cells, variants and mutants whose strain preservation number is CGMCC No.2487 and the enzyme and taxane produced thereof, specifically , involving a 7-xylose group that can hydrolyze the 7-xylose taxane in the wheat bran medium, and can also hydrolyze the 13- Enterobacter cloacae with side chains, and a method for preparing taxanes using Enterobacter cloacae. Background technique: [0002] Paclitaxel is a diterpene alkaloid with unique anticancer activity isolated from the bark of Taxus brevifolia by Wani et al. in 1971. Its chemical structural formula is: [0003] [0004] Since paclitaxel was approved by the FDA in 1992 for the treatment of ovarian cancer and metastatic breast cancer, it has been approved for marketing in more than 40 countries. In addition, it has been found that they also have g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P17/02C12R1/01
Inventor 戴均贵巢雄辉李建华李健丁志坚方唯硕
Owner INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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