Method for separating and extracting Sirolimus from fermentation liquid

A technology for sirolimus and sirolimus bacteria, which is applied in the field of rapid separation and extraction of sirolimus, can solve the problems of high production cost, low fermentation level of strains, complicated production and extraction routes, etc. The effect of amplification, simple separation route and high yield

Inactive Publication Date: 2011-05-25
SOUTHWEST UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the main key problems affecting the industrialization of sirolimus are the low level of strain fermentation and the complicated production and extraction routes. Due to the phenomenon of multiple impurity components and isomers in the fermentation process of rapamycin, sirolimus is currently The MOS extraction process is multi-step organic solvent extraction, adsorption, column chromatography and other steps, and the extraction yield is about 40-50%, resulting in high production costs. Therefore, to explore efficient separation and extraction technology and establish a new extraction process for Western Rolimus production has urgent practical significance

Method used

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  • Method for separating and extracting Sirolimus from fermentation liquid
  • Method for separating and extracting Sirolimus from fermentation liquid
  • Method for separating and extracting Sirolimus from fermentation liquid

Examples

Experimental program
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Effect test

Embodiment 1

[0017] 1. Sirolimus fermented mycelium: take sirolimus fermented liquid and filter it with a frame filter, remove the fermentation supernatant, and obtain sirolimus mycelium.

[0018] 2. Extraction and decolorization: the mycelium was extracted 3 times with acetone solvent, the extract was further extracted 2 times with ethyl acetate, decolorized with activated carbon, concentrated under reduced pressure to obtain the crude extract of sirolimus.

[0019] 3. High-speed countercurrent chromatographic separation: the high-speed countercurrent chromatograph adopts TBE-300A produced by Shanghai Tongtian Biotechnology Co., Ltd. The separation method is to mix n-hexane-ethyl acetate-ethanol-water in the ratio (7:5:5:5, v / v) to form a two-phase solvent system, the upper phase is the stationary phase, and the lower phase is the mobile phase. 2 mL / min, rotation speed 800 r / min, detection wavelength 280 nm, about 100 mg of sirolimus crude extract was dissolved in 10 mL lower phase, injec...

Embodiment 2

[0022] 1. Sirolimus fermentation mycelia: take the sirolimus fermentation broth and separate it with a centrifuge at 4000 r / min, remove the fermentation supernatant, and obtain sirolimus mycelia.

[0023] 2. Extraction and decolorization: the mycelium was extracted 3 times with methanol solvent, the extract was further extracted 2 times with ethyl acetate, decolorized with activated carbon, concentrated under reduced pressure to obtain the crude extract of sirolimus.

[0024] 3. High-speed countercurrent chromatographic separation: the high-speed countercurrent chromatograph adopts TBE-300A produced by Shanghai Tongtian Biotechnology Co., Ltd. The separation method is to mix n-hexane-ethyl acetate-ethanol-water (6:5:5:5, v / v) into a two-phase solvent system, the upper phase is the stationary phase, and the lower phase is the mobile phase. mL / min, rotation speed 900 r / min, detection wavelength 280 nm, about 100 mg of sirolimus crude extract was dissolved in 10 mL of the lower p...

Embodiment 3

[0027] 1. Sirolimus fermented mycelium: take sirolimus fermented liquid and separate it by frame filtration, remove the fermentation supernatant to obtain sirolimus mycelium.

[0028] 2. Extraction and decolorization: the mycelium was extracted 3 times with ethanol solvent, the extract was further extracted 2 times with ethyl acetate, decolorized with activated carbon, concentrated under reduced pressure to obtain the crude extract of sirolimus.

[0029] 3. High-speed countercurrent chromatographic separation: the high-speed countercurrent chromatograph adopts TBE-300A produced by Shanghai Tongtian Biotechnology Co., Ltd. The separation method is to mix n-hexane-ethyl acetate-ethanol-water (5:5:5:5, v / v) into a two-phase solvent system, the upper phase is the stationary phase, and the lower phase is the mobile phase. mL / min, rotation speed 900 r / min, detection wavelength 280 nm, about 100 mg of sirolimus crude extract was dissolved in 10 mL of the lower phase, and the sample w...

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Abstract

The invention provides a method for separating and extracting Sirolimus from fermentation liquid. The extraction process comprises the following steps of: preparing Sirolimus fermentation mycelia; performing extraction and decoloration; performing high-speed countercurrent chromatography separation; and drying. In the method, the multi-chromatography steps in the traditional extraction process are replaced by countercurrent chromatography separation technology; extraction is combined with countercurrent; and a Rapamycin product with purity of near 90 percent can be obtained by one-step countercurrent separation. Compared with the relatively complex traditional Rapamycin extraction process, the fermentation mycelia crude extraction and high-speed countercurrent chromatography method has the advantages of simple separation route, contribution to amplification of the process and suitability for quickly separating and preparing high-additional-value Rapamycin monomers from the fermentation liquid in large scale. The method for separating and extracting the Sirolimus from the fermentation liquid has the advantages of high yield, simple extraction steps, solvent saving and the like, and can be applied to industrial actual amplified production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for rapidly separating and extracting sirolimus from a fermented liquid. Background technique [0002] Sirolimus is a macrocyclic triene lactone-containing compound with a molecular structure such as (I), produced by fermentation of Streptomyces hygroscopicus, and is a new type of potent immunosuppressant with antifungal, antiproliferative and antitumor activities. [0003] [0004] Because it specifically binds and inhibits the mammalian target of rapamycin (mTOR), sirolimus has been used as an anti-rejection drug for organ transplantation and a drug for the treatment of autoimmune diseases. The derivatives Temsirolimus (temsirolimus), Everolimus (everolimus), and Deforolimus (AP23573, MK-8669) developed on the basis of sirolimus are now used as targeted, low-toxic anti-tumor cancer drugs into the clinic. [0005] At present, the main key problems affecting...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D498/18
Inventor 邹祥李进
Owner SOUTHWEST UNIV
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