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Preparation method of alpha-ketoglutarate

A kind of technology of ketoglutaric acid and glutamic acid

Active Publication Date: 2018-09-04
HENAN JULONG BIOLOGICAL ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, biological fermentation also has its disadvantages: long production cycle, low yield, mixing of various components in the product and fermentation broth, resulting in complex extraction and refining processes and high total cost

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0018] A kind of preparation method of α-ketoglutaric acid, concrete steps are as follows:

[0019] Inoculate the Escherichia coli strain on the seed medium, place it in a biochemical incubator and incubate it at 37°C for 20 hours, then apply gradient dilution on the complete medium (LB), pick single colonies and inoculate them on the seed medium respectively, and biochemically Incubate at 30-36°C for about 12 hours in an incubator. Inoculate into a 500mL seed bottle, culture conditions: temperature 30~36℃, shaker speed 200rmp, culture time 6~15h; when the OD660nm value grows to 5.0~15.0, a seed liquid is formed. Inoculate the seed solution into a 500mL fermentation bottle with fermentation medium, the inoculation amount: 5-15% of the volume of the fermentation medium in the fermentation bottle; culture conditions: temperature 30-38°C, shaker speed 200-800rmp, tank pressure 0.01-0.15Mpa, air flow rate 1-8L / min, dissolved oxygen maintained at 10-30%; when OD660nm grows to 10-2...

Embodiment 1

[0023] Pick Escherichia coli colonies from the fresh slant medium and inoculate them into a 500mL seed bottle added with ampicillin sodium resistant seed medium for cultivation. 8h; when OD 660nm When the value grows to 5.0-15.0, the seed liquid is obtained. Put the seed liquid into a 5L fully automatic fermenter with 3L fermentation medium at 10% inoculum size for secondary seed culture. The culture conditions are: temperature 30~36℃, tank pressure 0.01~0.05Mpa, air flow 1~8L / min, the stirring speed is 200~800r / min, the dissolved oxygen is maintained at 10~30%, the pH value is controlled between 7.0~7.2, the cultivation time is 5-8h, and the secondary seed liquid is obtained. The secondary seed solution was put into a 30L fully automatic fermenter with 18L fermentation medium at 10% inoculum for acidogenic fermentation. Fermentation conditions: temperature 30~36℃, tank pressure 0.01~0.15Mpa, air flow 5~50L / min, stirring speed 200~800r / min, dissolved oxygen maintained at 10~...

Embodiment 2

[0027] The steps of seed solution preparation, fermentation and transformation were the same as in Example 2, except that the ampicillin sodium resistance was not added to the seed medium added in the seed bottle. The content of ketoglutaric acid in the conversion solution was detected by high performance liquid chromatography to be 42.87 g / L.

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Abstract

The invention relates to a preparation method of alpha-ketoglutarate and belongs to the technical field of biological engineering. The method comprises the steps of fermentation production: performingfermentation culture on escherichia coli till OD660nm grows to 10-20, adding lactose into a fermentation culture medium to continue inducing culture, performing centrifugation to collect wet thalli,forming a transformation system with L-glutamic acid or L-glutamate, manganese sulfate, catalase and the wet thalli, regulating a pH (potential of hydrogen) of the transformation system to 4-8, and performing transformation at the temperature of 20-40 DEG C, rotating speed of 100-400rpm and air volume of 10-20L / min for 20-30h to form a transformation solution, and extraction: performing filtration, purification, decoloration, concentration and crystallization on the transformation solution to form alpha-ketoglutarate. According to the method, fermentation and transformation conditions are mainly optimized; a production period is shortened; the yield of alpha-ketoglutarate is increased; and the method is suitable for industrial amplification production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a preparation method of α-ketoglutarate. Background technique [0002] α-Ketoglutaric acid (2-Ketoglutaric acid), also known as 2-oxoglutaric acid, is an important biomolecule, one of the important intermediates in the tricarboxylic acid cycle, and a transporter of nitrogen and Symbionts in molecular oxidation. It plays an important role in the metabolism of microbial cells and is also an important precursor for the synthesis of various amino acids and proteins. Used in nutritional enhancers, as ingredients of sports nutrition drinks, organic intermediates, biochemical reagents and supporting reagents for liver function testing, physical enhancement supplements, etc. In addition, α-ketoglutaric acid and arginine can prepare 1:1 and 1:2 sperm-ketone mixtures. [0003] The existing α-ketoglutaric acid production process is basically based on chemical synthesis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/50C12R1/19
CPCC12P7/50
Inventor 谢沛曹华杰刘帅王卫富
Owner HENAN JULONG BIOLOGICAL ENG CO LTD
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