A kind of method for preparing phenylpyruvate

A technology of phenylpyruvate and bacteria, applied in the biological field, can solve problems such as the influence of phenylpyruvate yield

Active Publication Date: 2021-07-06
TAIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A kind of method for preparing phenylpyruvate
  • A kind of method for preparing phenylpyruvate
  • A kind of method for preparing phenylpyruvate

Examples

Experimental program
Comparison scheme
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 phenylpyruvate. First, the transaminase gene ata is inserted into the pET28a plasmid to construct transaminase recombinant Escherichia coli cells, and then the E. coli cells expressing the transaminase gene are induced and expressed, and finally the large intestine expressing the transaminase gene is Bacillus thallus and L-phenylalanine carry out catalytic reaction to obtain α-phenylpyruvate; The concentration of the Escherichia coli thallus expressing transaminase gene is 1.5-2.5g / L, and the concentration of L-phenylalanine is 4-phenylalanine 8g / L. The method of the present invention adopts optimal induction conditions combined with optimal catalytic conditions, avoiding the influence of pH, temperature, time, substrate concentration and bacterial cell concentration on the yield of phenylpyruvate, and the substrate conversion rate reaches 91%. Obtain high-quality and high-yield phenylpyruvate.

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 Patents(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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