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Method for preparing phenylpyruvic acid

A technology of phenylpyruvate and phenylalanine, which is applied in the biological field and can solve problems such as the impact of phenylpyruvate yield

Active Publication Date: 2020-05-08
TAIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzyme conversion method is also affected by many factors such as pH, inhibitor, temperature, substrate concentration, etc., and these factors have a greater impact on the yield of phenylpyruvate.

Method used

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  • Method for preparing phenylpyruvic acid
  • Method for preparing phenylpyruvic acid
  • Method for preparing phenylpyruvic acid

Examples

Experimental program
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Effect test

Embodiment 1

[0040] A method for preparing phenylpyruvate, the steps are:

[0041] (1) Construction of transaminase recombinant Escherichia coli strain: extract the transaminase gene ata in the Arthrobacter sp. Insert the double digested product into the pET28a plasmid to obtain the pET28a plasmid linked with transaminase, named pET28a-ata, and finally transform pET28a-ata into BL21 competent cells to obtain transaminase recombinant E. coli cells, named BL21(DE3) / pET28a-ata;

[0042] The plasmid was extracted from BL21(DE3) / pET28a-ata and verified by electrophoresis and sequencing after double enzyme digestion. figure 1 and figure 2 It showed that the genome was successfully extracted and PCR amplified to transaminase gene. The plasmid extracted from BL21(DE3) / pET28a-ata was digested with NcoI and XhoI to obtain two bands of 996bp and 5230bp (such as image 3 shown), are the lengths of pET28a and the target gene, respectively, and the results show that the recombinant plasmid pET28a-...

Embodiment 2

[0048] A method for preparing phenylpyruvate, the steps are:

[0049] (1) Construction of transaminase recombinant Escherichia coli strain: extract the transaminase gene ata in the Arthrobacter sp. Insert the double digested product into the pET28a plasmid to obtain the pET28a plasmid linked with transaminase, named pET28a-ata, and finally transform pET28a-ata into BL21 competent cells to obtain transaminase recombinant E. coli cells, named BL21(DE3) / pET28a-ata;

[0050] (2) Induced expression: BL21(DE3) / pET28a-ata was cultured on a shaker at 37°C at a rate of 100rpm / min, when the cell OD 600 When the concentration is 0.5, add IPTG with a final concentration of 0.1mmol / L, induce the reaction at 30°C for 6h, centrifuge at 8000rpm / min for 3min, and collect the cells to obtain Escherichia coli cells expressing the transaminase gene.

[0051] (3) Catalytic reaction: Escherichia coli cells expressing transaminase gene, L-phenylalanine and α-ketoglutaric acid were added to PBS bu...

Embodiment 3

[0054] A method for preparing phenylpyruvate, the steps are:

[0055] (1) Construction of transaminase recombinant Escherichia coli strain: extract the transaminase gene ata in the Arthrobacter sp. Insert the double digested product into the pET28a plasmid to obtain the pET28a plasmid linked with transaminase, named pET28a-ata, and finally transform pET28a-ata into BL21 competent cells to obtain transaminase recombinant E. coli cells, named BL21(DE3) / pET28a-ata;

[0056] (2) Induced expression: BL21(DE3) / pET28a-ata was cultured on a shaker at 37°C at a speed of 500rpm / min, when the cell OD 600 When the concentration is 0.7, add IPTG with a final concentration of 0.8mmol / L, then induce the reaction at 22°C for 12h, centrifuge at 8000rpm / min for 3min, and collect the cells to obtain Escherichia coli cells expressing the transaminase gene.

[0057] (3) Catalytic reaction: Escherichia coli cells expressing transaminase gene, L-phenylalanine and α-ketoglutaric acid were added to...

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Abstract

The invention discloses a method for preparing phenylpyruvic acid. The method comprises the following steps: firstly, inserting a transaminase gene ata into a pET28a plasmid to construct a transaminase recombinant escherichia coli bacterium, secondly, performing induction expression so as to obtain an escherichia coli bacterium expressing a transaminase gene, and finally performing a catalysis reaction on the escherichia coli bacterium expressing the transaminase gene and L-phenylalanine, so as to obtain alpha-phenylpyruvic acid, wherein the concentration of the escherichia coli bacterium expressing the transaminase gene is 1.5-2.5g / L, and the concentration of the L-phenylalanine is 4-8g / L. By adopting the method, optimal induction conditions are combined with optimal catalysis conditions,the influence of multiple aspects such as pH values, temperatures, time, substrate concentrations and bacterium concentrations upon the yield of the phenylpyruvic acid can be avoided, the substrate conversion rate is up to 91%, and phenylpyruvic acid of high quality and high yield can be obtained.

Description

technical field [0001] The invention relates to the field of biology, and more specifically, relates to a method for preparing phenylpyruvate. Background technique [0002] α-Phenylpyruvate (PPA) is one of the dicarbonyl compounds and is a precursor compound for the production of L-phenylalanine. In the fields of medicine and organic synthesis, phenylpyruvate is a very important intermediate. In addition, phenylpyruvate has important applications in food production and food additives. Recently, with the large-scale production of low-calorie sweetener aspendipeptide in Europe, the United States, Japan and other countries, the world's annual output has exceeded 10,000 tons. As its limiting production raw material, phenylpyruvate is increasing its market demand year by year. Incrementally, thus stimulating the research on the synthesis process of phenylpyruvate at home and abroad, how to prepare a large amount of phenylpyruvate has become a hot topic for everyone. Currently u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/40C12N15/70C12N15/54C12R1/19
CPCC12N9/1096C12N15/70C12P7/40C12Y206/01
Inventor 尹龙飞付永前罗希郑伟龙张莹莹尹丰伟
Owner TAIZHOU UNIV
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