Method for detecting intracellular protein changes in process of acting on gluconobacter oxydans by glutathione

A technology of Gluconobacter oxydans and glutathione, which is applied in biochemical equipment and methods, measuring devices, biological tests, etc., can solve the problems of slow growth, low yield, and inability to provide favorable information, so as to improve yield and optimize Effects of the fermentation process

Active Publication Date: 2012-06-27
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Gluconobacter oxydans is used for the industrial fermentation of vitamin C. This fermentation is completed by the joint action of Gluconobacter oxydans and Bacillus megaterium. However, Gluconobacter oxydans is fermented alone, with slow growth and low yield
After adding glutathione, the growth of bacteria and the production of 2-keto-L-gulonic acid were significantly improved, but the mechanism of action is still unclear, and no favorable information can be provided for future molecular modification of Gluconobacter oxydans

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  • Method for detecting intracellular protein changes in process of acting on gluconobacter oxydans by glutathione
  • Method for detecting intracellular protein changes in process of acting on gluconobacter oxydans by glutathione
  • Method for detecting intracellular protein changes in process of acting on gluconobacter oxydans by glutathione

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Embodiment 1

[0040] A method for detecting intracellular protein changes in the process of glutathione acting on Gluconobacter oxidans, characterized in that it comprises the following steps:

[0041] (1) Determination of intracellular protein:

[0042] ① Cell collection and quenching:

[0043]Take 3 parts of Gluconobacter oxydans and inoculate them into fermentation medium A respectively with a volume ratio of 8%, and inoculate another 3 parts of Gluconobacter oxidans into fermentation medium B with a volume ratio of 8%. The culture medium after inoculation was cultured and fermented at 28°C with a rotational speed of 200rpm. During the fermentation process, 1h and 15h were selected as sampling points to take out samples of the fermentation broth, and at 4°C, centrifuged at a rotational speed of 4000rpm to collect the cells in the lower layer and use Wash with phosphate buffer solution with a pH of 7.2, and immediately quench with liquid nitrogen to terminate the metabolic reaction; grin...

Embodiment 2

[0077] A method for detecting intracellular protein changes in the process of glutathione acting on Gluconobacter oxidans, comprising the steps of:

[0078] (1) Determination of intracellular protein:

[0079] ① Cell collection and quenching:

[0080] Get 4 parts of Gluconobacter oxidans and inoculate them into fermentation medium A respectively with a volume ratio of 10%, and inoculate another 4 parts of the Gluconobacter oxidans into fermentation medium B with a volume ratio of 10%. The inoculated culture medium was cultured and fermented at 30°C with a rotational speed of 220rpm. During the fermentation process, 1h and 15h were selected as sampling points to take out samples of the fermentation broth, and at 4°C, centrifuged at a rotational speed of 5000rpm to collect the cells in the lower layer and use Phosphate buffer solution with a pH of 7.3 was washed, quenched with liquid nitrogen, and the metabolic reaction was terminated; ground with liquid nitrogen to break the c...

Embodiment 3

[0103] A method for detecting intracellular protein changes in the process of glutathione acting on Gluconobacter oxidans, comprising the steps of:

[0104] ① Cell collection and quenching:

[0105] Get 5 parts of Gluconobacter oxydans and inoculate them into fermentation medium A respectively with a volume ratio of 16%, and inoculate another 5 parts of the Gluconobacter oxidans into fermentation medium B with a volume ratio of 16%. The inoculated culture medium was cultured and fermented at 32°C with a rotation speed of 250rpm. During the fermentation process, 1h and 15h were selected as sampling points to take out the fermentation broth samples, and the cells in the lower layer were collected by centrifugation at 4°C at a speed of 6000rpm. Wash with phosphate buffer solution with pH 7.4, quench with liquid nitrogen, and terminate the metabolic reaction; grind and break the cells with liquid nitrogen to obtain 5 parts of broken cells collected from medium A for 1 hour of ferm...

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Abstract

The invention discloses a method for detecting intracellular protein changes in a process of acting on gluconobacter oxydans by glutathione. The method comprises the following steps of: 1, determination of an intracellular protein: (1) collecting and quenching the cell, (2) extracting the intracellular protein, (3) determining protein concentration, (4) precipitating the protein, (5) subjecting the protein to reduction and enzymolysis, (6) labeling the protein, (7) mixing solutions, (8) identifying the protein through differential expression; 2, main component analysis; and 3, process analysis. By utilization of the method disclosed by the invention, the important proteins in a fermentation process can be found out by revealing the change rule of the intracellular protein after the gluconobacter oxydans is acted by the glutathione, the change rule of the content of the proteins provides a basis for learning the internal mechanism of the fermentation process, so that the fermentation process is further optimized, the yield of Vitamin C is increased, and a theoretic basis is provided for carrying out molecular modification on the gluconobacter oxydans. Meanwhile, new concept and method are provided for researching the industrial mixed fermentation process.

Description

technical field [0001] The invention belongs to the field of industrial microbes, and relates to a method for detecting intracellular protein changes in the process of glutathione promoting the production of 2-keto-L-gulonic acid by Gluconobacter oxidans. Background technique [0002] Protein is an important part of cells, and the study of protein structure and function will directly clarify the mechanism of changes in cells under different growth conditions. On the one hand, it can verify its upstream genomics information and provide the basis for subsequent genetic modification; on the other hand, it can provide guidance for its downstream metabolite research. The development of proteomics mainly relies on efficient protein separation and identification technology. There are mainly two separation methods: two-dimensional gel electrophoresis and liquid chromatography separation. The advantage of resolution is the mainstream of current proteomics development. Based on liqu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N27/62C12Q1/02
Inventor 元英进马倩张璐
Owner TIANJIN UNIV
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