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Method for producing poly-N-acetylneuraminic acid by microbial fermentation and purification method thereof

A technology of acetylneuraminic acid and microbial fermentation method, applied in the field of bioengineering, can solve the problems of high cost, unable to meet the requirements of various indicators, limiting the application of poly-N-acetylneuraminic acid, etc. quantity and difficulty, and the effect of reducing production costs

Inactive Publication Date: 2013-10-23
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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  • Abstract
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  • Application Information

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Problems solved by technology

Fermentative production of PSA has been reported in the literature such as Guo Liangdong et al. (screening of strains producing polysialic acid and acid production conditions, 1998), but various indicators of the pilot test can not meet the requirements, and its highest fermentation level is only It is about 1.2g / L, and the source of fermentation is sorbitol, the cost is relatively expensive, and endotoxin must be removed
Therefore, the therapeutic application of poly-N-acetylneuraminic acid (PSA) is limited

Method used

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  • Method for producing poly-N-acetylneuraminic acid by microbial fermentation and purification method thereof
  • Method for producing poly-N-acetylneuraminic acid by microbial fermentation and purification method thereof
  • Method for producing poly-N-acetylneuraminic acid by microbial fermentation and purification method thereof

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Experimental program
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Effect test

Embodiment 1

[0047] Embodiment 1 microbial fermentation method produces poly-N-acetylneuraminic acid

[0048]The fermentation strain is Escherichia coli CGMCC NO.5585, which was screened by mutagenesis in the Industrial Microbiology Laboratory of the Institute of Microbiology, Chinese Academy of Sciences. Seed tanks are fermented in 5 liters. The basal medium used is glucose 25g / L, ammonium sulfate 5g / L, casein peptone 15g / L, dipotassium hydrogen phosphate 20g / L, magnesium sulfate 0.4g / L, after mixing evenly, steam sterilization. After reaching 121°C, maintain the tank pressure at 0.09MPa and sterilize for 30 minutes. The sterilization process requires continuous stirring at 200rpm. After cooling, inoculate 9 liters of bacterial culture solution. Cultivate at 37°C for 12 hours to obtain seed culture solution.

[0049] The fermenter adopts 200 liters of fermentation. The basal medium is glucose 25g / L, ammonium sulfate 5g / L, casein peptone 15g / L, dipotassium hydrogen phosphate 20g / L, ma...

Embodiment 2

[0051] The separation and purification of embodiment 2 poly-N-acetylneuraminic acid

[0052] The fermentation broth obtained in Example 1 was used. Carry out the separation and purification of poly-N-acetylneuraminic acid according to the following steps:

[0053] 1. Separation of bacteria liquid

[0054] The 0.1 micron ceramic membrane tangential microfiltration method is used to separate the bacteria (microfiltration device) and the fermentation liquid. The filtrate volume is 7 / 8 of the original volume. Then wash the thalline with 3 / 8 of the original volume of water, microfilter the thalline washing liquid, and combine with the aforementioned filtrate.

[0055] 2. Remove small molecule impurities

[0056] The supernatant separated from step 2 was ultrafiltered with a 70,000 Dalton ultrafilter to concentrate macromolecules. The filtered waste was discarded. This step can concentrate the above supernatant at least 20 times the volume. The concentrated solution was washe...

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Abstract

The invention provides a method for producing poly-N-acetylneuraminic acid by fermentation of escherichia coli CGMCC NO.5585 and a purification method of the poly-N-acetylneuraminic acid. The high-purity poly-N-acetylneuraminic acid without endotoxins is further obtained and can meet the production requirements of foods, cosmetics and medicaments. According to the methods provided by the invention, the fermentation raw material uses cheap glucose to replace more expensive sorbitol, the yield of 5-6g / L can be still obtained during the fermentation stage, and the methods are particularly suitable for industrial fermentation production of PSA (polysialic acid); and finally, through the follow-up purification step, the high-purity PSA with the polymerization degree of at least above 70000Da, the purity of at least more than 95% (HPLC(high performance liquid chromatography)) and the yield of 4-4.5g / L can be obtained.

Description

technical field [0001] The invention relates to a method for producing poly-N-acetylneuraminic acid by using a microbial fermentation method and a purification method thereof, belonging to the technical field of bioengineering. Background technique [0002] There have been many studies and attempts on the production of poly-N-acetylneuraminic acid (PSA) by microbial fermentation. It can be done by using the natural strain of Escherichia coli. PSA itself has multiple uses such as drug sustained-release agent, immune adjuvant, and drug precursor. Moreover, further hydrolysis can obtain monomer N-acetylneuraminic acid (Neu5Ac), which is an important food and pharmaceutical raw material. Fermentative production of PSA has been reported in the literature such as Guo Liangdong et al. (screening of strains producing polysialic acid and acid production conditions, 1998), but various indicators of the pilot test can not meet the requirements, and its highest fermentation level is o...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P19/04C08B37/00C12R1/19
Inventor 晏礼明杨勇傅深展钱世钧张国清赵娟董黎爱毋鸿江
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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