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Method for extracting intracellular coenzymes A and organic acids from saccharopolyspora erythraea

A technology of saccharopolyspora rubrum and organic acid, which is applied in the field of bioengineering and can solve the problems of intracellular metabolite analysis and research without saccharopolyspora rubrum, cell wall thickness, small cell diameter, etc.

Active Publication Date: 2017-12-12
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Although researchers have studied the extraction and analysis methods of intracellular metabolites of different microorganisms, there is no report on the analysis and research of intracellular metabolites of Saccharopolyspora rubrum
At the same time, because Saccharopolyspora rubrum belongs to actinomycetes, its cell diameter is small, the cell wall is relatively thick, and the extraction of intracellular metabolites is difficult. The method for extracting intracellular metabolites of other microorganisms cannot be directly applied to red sugar Extraction of Intracellular Metabolites of Polyspora

Method used

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  • Method for extracting intracellular coenzymes A and organic acids from saccharopolyspora erythraea
  • Method for extracting intracellular coenzymes A and organic acids from saccharopolyspora erythraea
  • Method for extracting intracellular coenzymes A and organic acids from saccharopolyspora erythraea

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Experimental program
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Effect test

Embodiment 1

[0083] Embodiment 1, bacterial cell culture

[0084] 1. Strains and cultivation

[0085] (1) Strains

[0086] Saccharopolyspora erythraea E3 strain. Preservation method: Store the strain in 50% glycerol at -20°C.

[0087] (2) culture medium

[0088] Incline medium:

[0089]

[0090] Shake flask medium (g / L)

[0091]

[0092]

[0093] (3) Culture conditions

[0094] Incline culture

[0095] Use test tubes for slant culture. Spread the spores in the glycerol tube evenly on the slant medium with a bamboo stick, and culture at 34°C for 10 days.

[0096] shake flask culture

[0097] Take 20ml of sterile water and pour it into an inclined test tube, scrape off the spores on the inclined surface with a bamboo stick, pour the suspension into a 250ml shaker flask containing 5 small glass beads, and shake vigorously for 1min. Take 1ml of the spore suspension and add it to a 500ml shake flask containing 50ml shake flask culture medium. Put the shaker flask into a shak...

Embodiment 2

[0102] Embodiment 2, thalline inactivation and intracellular metabolite extraction

[0103] Coenzyme A substances and organic acids were obtained from Saccharopolyspora rubrum intracellularly by inactivation of bacteria and extraction of intracellular extracts.

[0104] 1. Inactivation method

[0105] The present inventors have found in research that when extracting organic acids from Saccharopolyspora rubrum, since the organic acid exists both inside and outside the cells of Saccharopolyspora rubrum, the cells must be cleaned after inactivation to remove the extracellular Effect of organic acids on determination of intracellular organic acid concentrations. Coenzyme A substances only exist in the cell, not outside the cell, and it is not necessary to remove the medium during the extraction process. Therefore, in the liquid nitrogen inactivation process, different methods of operation were adopted for the extraction of coenzyme A substances and organic acids.

[0106] Liqui...

Embodiment 3

[0130] Embodiment 3, intracellular metabolite extraction method comparison

[0131] After comparison, the inventor has focused on the following inactivation methods: liquid nitrogen inactivation and cold methanol inactivation; meanwhile, has focused on the following extraction methods: boiling ethanol method, liquid nitrogen freeze-thaw method and liquid nitrogen grinding method , to compare the advantages and disadvantages of these methods for the extraction effect of Saccharopolyspora rubrum intracellular extract.

[0132] 1. Analysis for the extraction and determination of intracellular coenzymes

[0133] The present inventors aimed at the four combinations of liquid nitrogen inactivation + liquid nitrogen freeze-thaw, liquid nitrogen inactivation + liquid nitrogen grinding, cold methanol inactivation + liquid nitrogen grinding, liquid nitrogen inactivation + boiling ethanol extraction, and the coenzyme A substances The performance of different extraction methods was compa...

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Abstract

The invention relates to a method for extracting intracellular coenzymes A and organic acids from saccharopolyspora erythraea. The inventor discloses a method suitable for extracting intracellular coenzyme A substances and organic acids from saccharopolyspora erythraea by comparison and analysis realized by using a method for extracting various intracellular metabolites, so that the basis can be provided for analyzing the metabolic characteristic in a synthesis process of erythrocin from the metabolic point of view.

Description

technical field [0001] The invention belongs to the field of bioengineering, more specifically, the invention relates to a method for efficiently extracting intracellular coenzyme A and organic acids of Saccharopolyspora rubrum. Background technique [0002] Saccharopolyspora rubrum is a Gram-positive filamentous bacterium. It is used in the production of erythromycin A, a broad-spectrum macrocyclic antibiotic. The antibacterial spectrum of erythromycin A is similar to that of penicillin, and it can be used as a backup drug for patients allergic to penicillin. In addition, erythromycin can also be used to synthesize a series of derivative drugs, which have functions of anti-parasite, anti-tumor, immunosuppression, anti-inflammation and treatment of gastrointestinal diseases. Because erythromycin A has great application value, people have done a lot of research on its production strain Saccharopolyspora rubrum. [0003] Erythromycin A molecule is composed of a 14-membered ...

Claims

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Application Information

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IPC IPC(8): C07H19/207C07H1/08C07C51/42C07C59/19C07C57/15C07C55/10C07C59/185C07C59/245C07C59/265
CPCC07C51/42C07H1/08C07H19/207C07C59/19C07C57/15C07C55/10C07C59/185C07C59/245C07C59/265
Inventor 储炬洪铭黄明志庄英萍陈冲冲牟翰庄振栋刘兆鹏陈国枝
Owner EAST CHINA UNIV OF SCI & TECH
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