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Method for producing alpha-ketoglutaric acid by virtue of enzymic method

A ketoglutarate and enzymatic technology, which is applied in the field of enzymatic production of α-ketoglutarate, can solve the problems of high fermentation cost of L-glutamate oxidase, difficult separation and purification of fermentation products, etc., and achieves easy separation. , The effect of reducing separation cost and cheap price

Inactive Publication Date: 2014-11-19
洛阳华荣生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to solve the deficiencies of the above-mentioned technical problems, to provide a method for enzymatically producing α-ketoglutarate, using glutamic acid racemase to convert L-glutamic acid into D-glutamic acid, and then using The highly active D-amino acid oxidase further converts the generated D-glutamic acid into α-ketoglutaric acid, thereby effectively avoiding the problem of high fermentation cost of L-glutamic acid oxidase and avoiding the fermentation The defect of difficult separation and purification of the product of the method is beneficial to industrial production

Method used

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  • Method for producing alpha-ketoglutaric acid by virtue of enzymic method

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Effect test

Embodiment 1

[0066] Step 1, construction of genetically engineered bacteria expressing glutamate racemase and D-amino acid oxidase simultaneously

[0067] (1), construction of expression vector pET24a-glr

[0068] a. According to the global public database GenBank, the record of accession number AB003685.1 is derived from the glutamic acid racemase encoded by Bacillus subtilis IFO 3336 glr Gene sequence, plus enzyme cutting sites at both ends Nde I and Bam HI, after the start codon was changed from TTG to ATG, the whole gene synthesis was carried out, and the synthetic glr The specific sequence of the gene is shown as SEQ ID NO: 1;

[0069] b. will be synthesized glr Genetic use Nde I and Bam HI double enzyme digestion, purification for later use;

[0070] c. Extract the Escherichia coli expression vector pET24a, use Nde I and Bam HI double enzyme digestion, and purified in the previous step glr The gene fragments were connected and transformed into Escherichia coli DH5α co...

Embodiment 2

[0089] Step 1, construction of genetically engineered bacteria expressing glutamate racemase and D-amino acid oxidase simultaneously

[0090] (1), construction of expression vector pET24a-glr

[0091] a. According to the global public database GenBank, the record of accession number AB003685.1 is derived from the glutamic acid racemase encoded by Bacillus subtilis IFO 3336 glr Gene sequence, plus enzyme cutting sites at both ends Nde I and Bam HI, after the start codon was changed from TTG to ATG, the whole gene synthesis was carried out, and the synthetic glr The specific sequence of the gene is shown as SEQ ID NO: 1;

[0092] b. will be synthesized glr Genetic use Nde I and Bam HI double enzyme digestion, purification for later use;

[0093] c. Extract the Escherichia coli expression vector pET24a, use Nde I and Bam HI double enzyme digestion, and purified in the previous step glr The gene fragments were connected and transformed into Escherichia coli DH5α co...

Embodiment 3

[0112] Step 1, construction of genetically engineered bacteria expressing glutamate racemase and D-amino acid oxidase simultaneously

[0113] (1), construction of expression vector pET24a-glr

[0114] a. According to the global public database GenBank, the record of accession number AB003685.1 is derived from the glutamic acid racemase encoded by Bacillus subtilis IFO 3336 glr Gene sequence, plus enzyme cutting sites at both ends Nde I and Bam HI, after the start codon was changed from TTG to ATG, the whole gene synthesis was carried out, and the synthetic glr The specific sequence of the gene is shown as SEQ ID NO: 1;

[0115] b. will be synthesized glr Genetic use Nde I and Bam HI double enzyme digestion, purification for later use;

[0116] c. Extract the Escherichia coli expression vector pET24a, use Nde I and Bam HI double enzyme digestion, and purified in the previous step glr The gene fragments were connected and transformed into Escherichia coli DH5α co...

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Abstract

The invention relates to a method for producing alpha-ketoglutaric acid by virtue of an enzymic method. The method comprises the following steps: respectively synthesizing a glr gene and a daao gene and constructing expression vectors pET24a-glr and pET24a-daao; after double digestion of the expression vectors pET24a-glr and pET24a-daao respectively, connecting the expression vectors pET24a-glr and pET24a-daao to construct a double expression vector pET24a-glr-daao and transforming the double expression vector to escherichia coli to prepare a genetically engineered bacterium which simultaneously expresses glutamate racemase and D-amino acid oxidase; adding a wet thallus of the genetically engineered bacterium into water in a fermentation tank, then adding L-glutamic acid and catalase, and finally adjusting the pH by virtue of sodium hydroxide to prepare a reaction system for reaction; separating and purifying alpha-ketoglutaric acid converted in the reaction system after reaction. The method provided by the invention is low in cost, high in conversion rate (over 84%) and product concentration, and suitable for industrial application.

Description

technical field [0001] The present invention relates to a method for producing α-ketoglutarate, in particular to a method for producing α-ketoglutarate by enzymatic method. Background technique [0002] α-Ketoglutaric acid (α-Ketoglutaric acid) is a key substance in cellular glucose metabolism, and participates in many cellular biochemical metabolic pathways, including tricarboxylic acid cycle, amino acid and protein metabolism, etc. α-Ketoglutarate is widely used in industry, it can be used as a dietary supplement, a component for the synthesis of some heterocyclic compounds, as a protective agent against oxidative stress in humans and animals, and in combination with other substances can improve athlete performance and promote rehabilitation etc. [0003] There are many ways to produce α-ketoglutarate, such as chemical synthesis, microbial fermentation, enzymatic conversion and so on. The chemical synthesis method uses succinic acid and diethyl oxalate as raw materials ...

Claims

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Application Information

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IPC IPC(8): C12P7/50C12N1/21C12N15/70C12R1/19
Inventor 范文超丁鹏化春光
Owner 洛阳华荣生物技术有限公司
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