Production method for alpha-ketoglutaric acid

A technology of ketoglutaric acid and production method, applied in the direction of introducing foreign genetic material by using carrier, recombinant DNA technology, fermentation, etc., which can solve the problems of high fermentation cost of L-glutamic acid oxidase, difficulty in separation and purification of fermentation products, etc. , to achieve the effect of easy separation, reduced separation cost and low price

Inactive Publication Date: 2014-11-05
洛阳华荣生物技术有限公司
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  • 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 production method of α-ketoglutarate, which uses glutamate racemase to convert L-glutamic acid into D-glutamic acid, and then utilizes the highly active The D-amino acid oxidase can further convert the generated D-glutamic acid into α-ketoglutarate, thus effectively avoiding the problem of high fermentation cost of L-glutamic acid oxidase, and also avoiding the production of fermentation products The defect of difficult separation and purification is beneficial to industrial production

Method used

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  • Production method for alpha-ketoglutaric acid

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Embodiment 1

[0057] Step 1, construction of glutamic acid racemase genetically engineered bacteria

[0058] (1), according to GenBank: AB003685.1 recorded from Bacillus subtilis IFO 3336 encoding glutamic acid racemase 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;

[0059] (2), the synthesized glr Genetic use Nde I and Bam HI double enzyme digestion, purification for later use;

[0060] (3), extract 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 competent cells;

[0061] (4) After picking positive transformants and identifying them by sequencing, the plasmids were extracted to obtain glr Expression vector pET24a-glr;

...

Embodiment 2

[0075] Step 1, construction of glutamic acid racemase genetically engineered bacteria

[0076] (1), according to GenBank: AB003685.1 recorded from Bacillus subtilis IFO 3336 encoding glutamic acid racemase 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;

[0077] (2), the synthesized glr Genetic use Nde I and Bam HI double enzyme digestion, purification for later use;

[0078] (3), extract 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 competent cells;

[0079] (4) After picking positive transformants and identifying them by sequencing, the plasmids were extracted to obtain glr Expression vector pET24a-glr;

...

Embodiment 3

[0093] Step 1, construction of glutamic acid racemase genetically engineered bacteria

[0094] (1), according to GenBank: AB003685.1 recorded from Bacillus subtilis IFO 3336 encoding glutamic acid racemase 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;

[0095] (2), the synthesized glr Genetic use Nde I and Bam HI double enzyme digestion, purification for later use;

[0096] (3), extract 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 competent cells;

[0097] (4) After picking positive transformants and identifying them by sequencing, the plasmids were extracted to obtain glr Expression vector pET24a-glr;

...

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Abstract

The invention relates to a production method for alpha-ketoglutaric acid. The method includes: synthesizing a glr gene with a specific sequence shown as SEQ ID NO:1 and a daao gene with a specific sequence shown as SEQ ID NO:2 respectively, and constructing a glutamate racemase genetic engineering bacterium and a D-amino acid oxidase genetic engineering bacterium; culturing the constructed bacteria, then collecting thalli; adding mixed wet thalli of the two bacteria into water in a fermentation tank, then adding L-glutamic acid to a final concentration of 90-100g / L, then adding catalase to a concentration of 1000000-4000000units / L, and finally adjusting the pH to 7.5-8.2 by sodium hydroxide so as to obtain a reaction system to perform reaction; a and at the end of the reaction, separating and purifying the alpha-ketoglutaric acid converted from the reaction system, thus obtaining the alpha-ketoglutaric acid. With a low cost, the method provided by the invention can achieve a high conversion rate and product concentration, with the conversion rate up to over 84%, and does not generate by-product, thus being more suitable for industrial application.

Description

technical field [0001] The invention relates to a method for producing α-ketoglutarate, in particular to a method for enzymatically producing α-ketoglutarate. 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 to prepare by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/50C12N15/70
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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