Fibrized cellulomonas cartae, hydrolase and application thereof in taxane conversion aspect

A cellulomonas and hydrolytic enzyme technology, applied in the field of biochemistry, can solve the problems of unsuitability for industrial production, reduced hydrolysis efficiency, and low microbial enzyme production

Inactive Publication Date: 2009-03-11
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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[0008] The enzyme production of microorganisms in the existing technology is generally low, and they are all intracellular enzymes, resulting in a decrease in hydrolysis efficiency. At the same time, the bacteria are easy to adsorb substrates and products, so they are not suitable for industrial production.

Method used

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  • Fibrized cellulomonas cartae, hydrolase and application thereof in taxane conversion aspect
  • Fibrized cellulomonas cartae, hydrolase and application thereof in taxane conversion aspect
  • Fibrized cellulomonas cartae, hydrolase and application thereof in taxane conversion aspect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Hydrolysis of 7-xylose-paclitaxel by Cellulomonas fibrosis CCTCC No.M 207130

[0039] A medium (pH 7.0 before sterilization) containing 1% glycerol, 0.2% peptone, 0.2% yeast extract, and 0.2% dipotassium hydrogenphosphate was used as a seed medium.

[0040] Distribute 20ml of the above-mentioned seed medium into 100ml conical flasks, sterilize at 120°C for 15min, inoculate 1 inoculation loop of the agar slant culture of Cellulomonas cellulosus XZ-5 strain into it, and inoculate it at 30°C Cultured with shaking for 2 days as a seed solution.

[0041] Put 100ml of 1% starch, 0.2% yeast extract, 0.2% ammonium chloride, 0.1% NaH into a 500ml Erlenmeyer culture bottle 2 PO 4 and 0.1% Na 2 HPO 4 Medium, pH7, autoclaved at 121°C for 30min. Inoculate 1ml of seed liquid into this medium, shake the flask at 150rpm, and culture at 30°C for 5 days. Centrifuge to collect the supernatant, which is the crude enzyme solution.

[0042] 1 ml of 50 mM potassium phosphate...

Embodiment 2

[0048] Example 2: Hydrolysis of 7-xylose-10-deacetyl-paclitaxel by Cellulomonas cellulosus CCTCC No.M 207130 and purification of the product

[0049] Add 10ml of 7-xylose-10-deacetyl-paclitaxel methanol solution (concentration: 5mg / ml) to 90ml of the crude enzyme solution described in Example 1, 50rpm, react at 30°C for 20 hours, then add 20ml of ethyl acetate Extraction, total extraction 3 times, combined upper organic phase, evaporated to dryness under reduced pressure, obtains 105mg of solid matter, goes up silica gel column (20g) and carries out elution with chloroform:methanol (98:2), obtains 40mg10-deacetylpaclitaxel (yield 91%).

Embodiment 3

[0050] Example 3: Hydrolysis of Cellulomonas cellulosus CCTCC No.M 207130 mixed taxane xylosides and purification of products

[0051] Put 500ml of 1% birch xylan, 0.5% urea, 0.2% KH in a 2000ml Erlenmeyer culture bottle 2 PO 4 and 0.1% K 2 HPO 4 Medium, pH 7, autoclaved for 30 min. 10ml of the seed solution described in Example 1 was inoculated in this medium, and cultured in shake flasks at 30° C. for 2 days at 200 rpm. The supernatant was collected by centrifugation and stored at -20°C for later use. Mix 200ml containing 7-xylose-10-deacetyltaxol, 7-xylose-10-deacetylcephalomannine, 7-xylose-10-deacetyltaxol C (from natural yew extract Separated, methanol solutions (5 mg / ml) with contents of 65%, 9.9%, and 3.3%) were added to 2000 ml of the crude enzyme solution as described above, 50 rpm, and reacted at 35° C. for 8 hours. The reaction solution was extracted three times with ethyl acetate, 200ml each time, the combined organic phases were evaporated to dryness and di...

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Abstract

The invention provides a fibration Cellulomonas spp and a hydrolytic enzyme thereof for effectively transforming taxane xyloside into taxol or the analogues thereof, and an effective new method for preparing the taxol or the analogues thereof through biotransformation reaction. The method can prepare the taxol or the analogues thereof with high efficiency, low cost and environmental protection, which provides an effective approach adapting to industrial production for making full use of taxane resources.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and specifically relates to a method for preparing paclitaxel and its analogues by using microorganisms that hydrolyze taxane xyloside. Background technique [0002] Taxanes are a class of diterpene compounds, among which paclitaxel (Paclitaxel, formula II) and docetaxel (Docetaxel) are widely used as first-line clinical drugs in the treatment of various tumors. At present, the main ways to produce paclitaxel are: (1) directly extracting paclitaxel from Taxus plants; (2) semi-synthetic method - extracting and separating compounds with taxol mother core structure from Taxus genus plants, and then using chemical methods Semi-synthetic paclitaxel, docetaxel, etc. Such compounds with paclitaxel core structure mainly include baccatin III and 10-deacetyl baccatin III (abbreviated as 10-DABIII). [0003] (Formula II) [0004] wherein Ph is phenyl, Ac is acetyl, and Bz is benzoyl. [0005] In...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12N1/20C12P17/02
CPCC12N9/2402C07D305/14C12Y302/01037C12P17/02C12N9/24C12R1/01C12N1/205C12R2001/01
Inventor 杨凌栾宏伟刘兴宝
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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