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Preparation method and application of <15>N-labeled bacterial protein

A bacterial protein and labeling technology, applied in the field of preparation of 15N-labeled bacterial protein, can solve the problems of enzyme digestion and mass spectrometry deviation, protein concentration error, etc., so as to overcome the growth of lag period, reduce cost, and eliminate enzyme digestion and mass spectrometry deviation. Effect

Pending Publication Date: 2020-03-06
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the traditional method, there are enzyme digestion and mass spectrometry deviations, and there is an error between the calculated protein concentration and the actual protein concentration

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] A sort of 15 The preparation method of N-labeled bacterial protein, comprises the steps:

[0041] Seed culture: inoculate the spore liquid into the seed medium, and cultivate it at 33-35°C for 22-26 hours at a speed of 120-180 rpm to obtain the seed liquid;

[0042] Pre-treatment: take an appropriate amount of the seed solution and place it in a centrifuge tube, centrifuge at 3500-4500 rpm for 4-8 minutes, wash with physiological saline, and obtain a seed treatment solution;

[0043] Marking culture: put the seed treatment solution in a shaker, add the fermentation medium solution, shake the flask for 24 hours of fermentation, then transfer it to a fermenter, and ferment for 45 to 50 hours. The method of feeding is used in the fermentation and cultivation project Adding the fermentation medium solution, terminating the fermentation when the carbon dioxide release rate (CER) in the fermentation system decreases, to obtain a fermentation broth;

[0044] Isolate protein: S...

Embodiment 2

[0047] A sort of 15 The preparation method of N-labeled bacterial protein, comprises the steps:

[0048] Seed culture: take 1.4mL spore count as 10 6 The spore liquid was inoculated into the seed bottle containing the seed medium, and cultivated at 34°C for 24 hours at a speed of 150rpm to obtain the seed liquid;

[0049] Pretreatment: get the described seed liquid of 4mL (0.033g) and place in 10mL centrifuge tube, centrifuge 5 minutes under 4000rpm, wash three times with saline, remove corn steep liquor, obtain seed treatment liquid, avoid the 14 The influence of N;

[0050] Marking culture: put the above seed treatment solution in a baffle shake flask filled with fermentation medium, and ferment the shake flask for 24 hours, then transfer it to a 1L fermenter, and add the fermentation medium by feeding during the fermentation process solution, fermented and cultivated for 48 hours to obtain a fermented liquid, and the fermented liquid had 587 mL;

[0051] Isolate protein...

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Abstract

The invention discloses a preparation method and application of <15>N-labeled bacterial protein. The preparation method comprises the following steps: seed culture: inoculating spore liquid into a seed culture medium, and carrying out culturing at the rotating speed of 120-180 revolutions per minute and under the temperature of 33-35 DEG C to obtain seed liquid; pretreatment: putting the seed liquid into a centrifuge tube, carrying out centrifuging, and carrying out washing with normal saline to obtain a seed treatment solution; marking culture: adding the seed treatment solution into a fermentation medium solution, shaking a flask for fermentation, then putting the flask into a fermentation tank for continuous fermentation culture, adding the fermentation medium solution in a material supplementing manner in the continuous fermentation process, and stopping fermentation to obtain fermentation liquid; and protein separation: carrying out suction filtration on the obtained fermentationliquid to obtain bacterial bodies, carrying out freezing with liquid nitrogen and grinding, and carrying out freeze drying to obtain the <15>N-labeled bacterial protein. According to the preparation method disclosed by the invention, the <15>N can be efficiently labeled to the bacterial protein to obtain the <15>N-labeled protein.

Description

technical field [0001] The invention relates to the technical field of isotope labeling, in particular to a 15 Preparation method and application of N-labeled bacterial protein. Background technique [0002] Aspergillus niger, Deuteromycotina, Hyphospora, Hyphospora, Polyporaceae, a common species in the genus Aspergillus, is widely distributed in grains, plant products and soils all over the world. Aspergillus niger is an important fermentation industrial strain, which can produce amylase, acid protease, cellulase, pectinase, glucose oxidase, citric acid, gluconic acid and gallic acid, etc. Some strains can also produce hydroxypregnant Ketones are converted to androstenes. The optimum temperature for growth is 37°C, and the minimum relative humidity is 88%, which can cause mildew in grains with high moisture content and mildew in other industrial equipment. Aspergillus niger can be used to produce saccharified feed and biological fertilizer. Aspergillus niger can also b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14G01N27/62C12R1/685
CPCC12N1/14G01N33/6848
Inventor 夏建业张薇刘鹏陈敏庄英萍
Owner EAST CHINA UNIV OF SCI & TECH
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