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Actinoplanessp. strain and its use in preparation of fidaxomicin

A technology of swimming actinomycetes and fidaxomicin, which is applied in the directions of bacteria, microorganism-based methods, biochemical equipment and methods, etc., to achieve the effects of easy separation and purification, single fermentation component and high fermentation unit

Active Publication Date: 2014-09-24
NCPC NEW DRUG RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

WO2007048059A2 is one of the few patent documents on fidaxomicin fermentation strains and fermentation methods, but its fermentation unit is only about 50mg / L

Method used

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  • Actinoplanessp. strain and its use in preparation of fidaxomicin
  • Actinoplanessp. strain and its use in preparation of fidaxomicin
  • Actinoplanessp. strain and its use in preparation of fidaxomicin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Isolation and identification of embodiment 1 bacterial strain

[0040] 1. Strain isolation

[0041] Agrimony samples were collected from Yunnan (23°22'39.648" north latitude, 104°47'17.0808" east longitude, 1498.7432m above sea level). The samples were stored at 4°C after collection, and the strains were isolated quickly.

[0042]Agrimony samples were rinsed with tap water several times to remove the surface soil, and then the following procedures were used for surface disinfection under sterile conditions: 0.01% Tween-20 treatment for 1 min, sodium hypochlorite with an available chlorine content of 4.5%-5% for 3 min, sterile Treat with 2.5% sodium thiosulfate for 10 minutes, 75% ethanol for 5 minutes, wash with sterile water 3-4 times, soak in sterile 10% sodium bicarbonate for 10 minutes. The surface-sterilized Agrimony samples were fully blotted dry with sterile filter paper, crushed under aseptic conditions and sprinkled on the separation medium plate, and cultivat...

Embodiment 250L

[0058] The preparation of embodiment 250L tank fermentation Fidaxomycin

[0059] (a) Preparation of Actinomycetes mobilis strain N12W0304 seed solution

[0060] The seed medium was inoculated with Actinomycetes mobilis strain N12W0304, and cultured at 32° C. at 220 rpm for 48 hours to obtain seed liquid.

[0061] The preparation method of the seed medium is: 40.0 grams of cornstarch, 4.0 grams of glucose, 8.0 grams of peptone, 5.0 grams of beef extract, 5.0 grams of yeast extract powder, add tap water to dissolve, dilute to 1000ml, pH value 7.0-7.2, extinguish at 121 °C Bacteria 30min.

[0062] (b) Preparation of fermented broth of actinomycetes

[0063] Inoculate the 50L fermentation tank with the fermentation medium with the inoculum amount of 10% by volume of the seed solution. The temperature of the tank is 28°C, the tank pressure is 0.05±0.01Mpa, the ventilation rate is 20L / min, stirring at 400rpm, and fermented for 120h.

[0064] The preparation method of the fermenta...

Embodiment 3

[0070] Embodiment 3 Refining Fidaxomycin

[0071] 51.8 g of the crude fidaxomicin prepared in Example 2 was dissolved in 400 ml of methanol, filtered through a 0.45 μm membrane, and separated by HPLC. Preparative column, Daisogel-C1830×250mm10μm; mobile phase, 57% acetonitrile water, containing 0.05% trifluoroacetic acid; flow rate 40ml / min; UV254nm detection. The effluents connected to the chromatographic peak of fidaxomicin (RT≈15~17min) were combined and concentrated under reduced pressure to obtain 26.6 g of the product—fine product of fidaxomicin, with a content of 97.0% and a total yield of 61.4%.

[0072] Product is identified, and its result is as follows:

[0073] (1) Appearance: The prepared product exists in the state of white powder, glassy transparent solid, and crystalline powder.

[0074] (2) Solubility: The prepared product is soluble in methanol, ethanol, chloroform, ethyl acetate, and difficult to use in water and n-hexane.

[0075] (3) UV spectrum

[007...

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Abstract

The invention discloses an Actinoplanessp. strain and its use in preparation of fidaxomicin. The Actinoplanessp. strain is entophytic Actinoplanessp. N12W0304, is preserved in the China general microbiological culture collection center (CGMCC) on December 26, 2012 and has a preservation number of CGMCC No.7043. A fidaxomicin production process comprises the following steps of preparing an Actinoplanessp. N12W0304 seed solution and fermentation broth, carrying out centrifugation of the fermentation broth to obtain mycelia, carrying out extraction, condensation, dissolution, filtration, resin adsorption and drying of the mycelia to obtain a fidaxomicin crystal crude product, dissolving the fidaxomicin crystal crude product, and carrying out liquid chromatographic separation and pressure-reduction drying of the solution to obtain a fidaxomicin refined product. Under the fermentation conditions, the Actinoplanessp. strain has a fidaxomicin fermentation unit which is more than 1400mg / L and is higher than the fidaxomicin fermentation yield reported at present. The fidaxomicin fermentation process provided by the invention is simple, is suitable for industrial production and realizes a total product yield of 60%.

Description

technical field [0001] The present invention relates to microorganisms and their application in the field of pharmacy. Specifically, it is actinomycetes mobilis and its application in the preparation of fidaxomicin. Background technique [0002] Fidaxamycin is a class of 18-membered ring macrolide antibiotics produced by the fermentation of actinomycetes. It mainly inhibits bacterial RNA polymerase to rapidly produce anti-clostridium difficile infection (CDI) effect. Its treatment of CDI is superior to existing drugs. The currently found fidaxomicin-producing bacteria are mainly Dactylosporangium sp. D. aurantiacum subsp. hamdenensis NRRL18085 strain and Actinoplanes sp.) Deccan Plateau Actinomycetes (A. deccanensis) ATCC21983 strain. At present, the research on fidaxomicin mainly involves the chemical structure, dosage form, crystal form, use and composition of fidaxomicin, such as CN200580048715.3, WO2009070779A1, USP7906489B2, USP7378508B2, USP7863249B2 and other paten...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P19/62C12R1/045
Inventor 路新华郑智慧丁彦博可爱兵穆云龙徐岩单越琦蔡超靖蔡喜田霍培元赵峰沈文斌马瑛段宝玲
Owner NCPC NEW DRUG RES & DEV
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